CN101168049A - Application of interleukin-22 in the preparation of medicines for treating liver diseases and its preparation method - Google Patents

Abstract

The invention discloses the use of interleukin-22 in the preparation of medicines for treating liver diseases, especially the application in the preparation of medicines for treating alcoholic liver diseases and non-alcoholic fatty liver diseases, which expands the application range of interleukin-22 and brings liver disease patients Gospel; the present invention also discloses a preparation method of human recombinant interleukin-22. The method is used to produce human recombinant interleukin-22 with high expression and low production cost, which provides a basis for large-scale production and application of interleukin-22.

Description

Application of interleukin-22 in the preparation of medicines for treating liver diseases and its preparation method

technical field

The present invention relates to the medical use and preparation method of interleukin-22, in particular to the use of interleukin-22 in the preparation of medicines for treating liver diseases, and also to the preparation method of recombinant human interleukin-22.

Background technique

Interleukin-22 (English name is Interleukin 22, abbreviated as: IL22) is a cytokine discovered in 2000, which is secreted by activated T cells and NK cells (Wolk K et al., J Immunol 2002; 168:5397～402) , exert biological effects through heterologous receptor complexes (IL22R1, IL10R2) (XieMH et al. J Biol Chem 2000; 275; 31335～9), IL10R2 is widely distributed, and the expression of IL22R1 is highly restricted, mainly expressed in the pancreas, skin , liver, lungs and kidneys. Neither resting nor activated immune cells expressed IL-22R1 (Wolk K et al., Immunity 2004;21:241-54). The medical uses of interleukin-22 mainly include: (1) in the treatment of hyperlipidemia, hypertriglyceridemia, obesity and/or diabetes (patent application number: 200510023103.0), (2) in the treatment of pancreatic dysfunction (US Patent No.: 7,226,591) There is currently no literature on the treatment of liver diseases with interleukin-22.

Alcoholic liver disease and nonalcoholic fatty liver disease are genetic-environment-metabolic stress-related diseases. Alcoholic liver disease and nonalcoholic fatty liver disease can coexist with viral hepatitis. In addition to alcoholic liver disease, nonalcoholic fatty liver disease can lead to liver disease-related disability and death, and it is also associated with type 2 diabetes, metabolic syndrome and other diseases. Related cardiovascular and cerebrovascular events are closely related (Brunt E: Nonalcoholic steatohepatitis. Semin Liver Dis 2004, 24: 3-20).

Alcoholic liver disease (ALD) refers to a series of diseases such as liver damage caused by long-term excessive drinking, and its pathological changes include alcoholic fatty liver, alcoholic hepatitis, alcoholic liver fibrosis, and alcoholic liver cirrhosis , Several pathological changes can occur alone or simultaneously. With the continuous improvement of people's living standards, the incidence of alcoholic liver disease has shown an obvious upward trend, and it has become the second largest liver disease after viral liver disease in our country.

At present, there is no effective drug for the treatment of ALD. The drugs currently used in clinical practice include glucocorticoids, PTX (pentoxifylline) and anti-TNF-α treatment. Glucocorticoids are considered to improve the symptoms of alcoholic hepatitis in the acute phase , improve the short-term survival rate, but it does not have a favorable effect on the prognosis, liver complications and improvement of liver histological morphology (Bergheim I et al., Dig Dis. 2005; 23(3-4): 275-84). PTX is used in the treatment of alcoholic hepatitis and cirrhosis because of its anti-inflammatory effect, effective prevention of hepatorenal syndrome and good safety.

Nonalcoholic fatty liver disease (NAFLD) is a clinical syndrome with liver histopathological changes similar to alcoholic liver disease but without a history of excessive alcohol consumption. The prevalence of NAFLD in the general population of Western Europe, the United States, and Japan is 10-24%, the prevalence of NASH (non-alcoholic steatohepatitis) is 2%-5%, and the prevalence of NAFLD in obese patients is as high as 57.5%-74%. With the development of society and economy, the prevalence of NAFLD has increased rapidly, and it has become one of the three major liver diseases that endanger human health.

NAFLD is a genetic-environmental-metabolic stress-related disease that is closely associated with insulin resistance and its associated metabolic syndrome and genetic susceptibility. NAFLD can not only lead to liver disease-related disability and death, but also is closely related to the high incidence of atherosclerotic cardiovascular and cerebrovascular events. There are no effective drugs for the treatment of NAFLD.

At present, there are two main methods for preparing IL22: (1) prokaryotic expression system (Ronaldo Alves Pinto Nagem et al., Crystal Structure of Recombinant Human Interleukin-22 Structure, Vol.10, 1051-1062, August, 2002), the prokaryotic expression system is the use of pET A series of vectors, the induction method is chemical induction (IPTG induced expression), and its disadvantage is high cost; (2) eukaryotic expression system (Jing Li et al., International Immunopharmacology 4 (2004) 693-708), using eukaryotic expression system to express hIL22 is soluble, and its disadvantage is that the expression level is small, which cannot meet the needs of animal experiments; therefore, it is necessary to find a preparation method with high expression level and low cost.

Contents of the invention

One of the objects of the present invention is to provide a use of interleukin-22.

Another object of the present invention is to provide a preparation method of recombinant human interleukin-22.

The invention provides the application of interleukin-22 in the preparation of medicines for treating liver diseases.

The interleukin-22 refers to human interleukin-22, recombinant human interleukin-22, mouse interleukin-22 or recombinant mouse interleukin-22 and the like.

The liver disease refers to one of alcoholic liver disease, non-alcoholic fatty liver disease, viral hepatitis, acute liver failure, liver fibrosis, liver ischemia-reperfusion injury, and failure after liver transplantation.

The aforementioned liver disease refers to alcoholic liver disease or non-alcoholic fatty liver disease.

The preparation method of recombinant human interleukin-22 of the present invention comprises the following steps:

(1) Utilize human fresh peripheral blood, cultivate human peripheral blood lymphocytes, and extract total RNA;

(2) Using total RNA as a template and using f1 and r1 as primers to perform RT-PCR amplification to obtain the cDNA sequence of hIL22; the sequences of the primers f1 and r1 are as follows:

f1: 5'GAGAATTCATATGGCACCCATCAGCTCCCCA3';

r1: 5′ACGGATCCTCAAATGCAGGCATTTCT 3′

Then use the obtained cDNA as a template and use f1 and r1 as primers to carry out PCR amplification to obtain the DNA sequence of hIL22;

(3) Digest the DNA sequence of hIL22 obtained in step (2) and the plasmid pBV220 with EcoRI and BamHI respectively; then mix the two, connect the two with T4 ligase, and then convert the ligated product by electroporation or CaCl ₂ The host cells were transformed by the method, screened and identified, and the recombinant plasmid pBV220/hIL22 was obtained;

(4) Transform the host cells with the recombinant plasmid pBV220/hIL22 by electroporation or _CaCl2 method; then inoculate on LB medium for culture, then centrifuge, resuspend the sediment, break up the bacteria by ultrasonic waves, and then denature the obtained inclusion bodies, The recombinant human interleukin-22 protein of the present invention is obtained by dissolving, purifying, refolding and concentrating.

The detailed operation steps of the above-mentioned preparation method of recombinant human interleukin-22 are as follows:

(1) Dilute human fresh peripheral blood twice with PBS solution containing 2mmol/L EDTA, spread it on an equal volume of lymphocyte separation medium, centrifuge at 700-1000rpm, and suck out the gray-white layer cells with a capillary glass tube, Wash the gray-white coat cells 2 to 3 times with Hanks solution, then suspend the gray-white coat cells in 1640 culture medium, and culture them in a 37°C, 5% CO ₂ incubator for 24 hours, then add ConA (2mg/ml) and anti- CD ₃ (4mg/ml) co-stimulatory cells were then cultured for 24 hours at 37°C in a 5% CO ₂ incubator, and total RNA was extracted with a total RNA extraction kit;

(2) Use total RNA as template, and use f1 and r1 as primers

f1: 5′GAGAATTCATATGGCACCCATCAGCTCCCCA3′

r1: 5′ACGGATCCTCAAATGCAGGCATTTCT3′

Perform RT-PCR amplification to obtain the cDNA sequence of human interleukin-22;

The ratio of each part of the RT-PCR reaction system is: total RNA, 5ul; RNase inhibitor; 0.5ul; Oligo (dT), 0.5ul; DEPC treated water, 6.5ul; a total of 12ul; RT: Add to the above system: 5×buffer 4ul, 2.5mMdNTP 2ul, AMV 1ul, RNase inhibitor 1ul, 42°C for 1h, 85°C for 5min to 4°C;

Then use the first-strand cDNA produced by reverse transcription as a template, and use f1 and r1 as primers to carry out a PCR reaction. The proportions of each part of the PCR reaction system are: 10PCR reaction × buffer 2.0ul, 2.0mmol/LdNTP 2.0ul, 5pmol/ul fl 2.0ul, 5pmol/ulrl2.0ul, reverse transcription product 4.0ul, TaqDNA polymerase 0.5ul, ddH2O 7.5ul, a total of 20ul; the PCR reaction conditions are 94°C pre-denaturation 3min, 94°C 30S, 50°C 30S, 72°C 30S, 30 cycles; 72°C extension for 7 minutes; use 1.2% agarose gel to separate PCR products;

(3) Add restriction endonucleases EcoRI and BamHI to the RT-PCR product and plasmid pBV220 respectively, digest in a water bath at 37°C for 3.5 hours, then mix the two at a ratio of 10-4:1, and add T4 ligase, Carry out the ligation reaction at 16°C for 12 hours, transform the ligated product into host cells by electroporation or CaCl ₂ method, screen recombinant clones by ampicillin resistance, extract corresponding plasmids, identify by enzyme digestion, and sequence to obtain recombinant plasmid pBV220/hIL22;

(4) Transform the recombinant plasmid pBV220/hIL22 into host cells by electroporation or _CaCl2 method; the transformed colony is activated overnight at 30°C, and inoculated in LB medium at a ratio of 1-2% by volume the next day, and cultured at 30°C for 2 ~3hr, then placed on a water-bath shaker at 42°C, cultured at 180-220rpm for 4-8hr; then centrifuged at 4°C, 10,000-12,000rpm for 10-15min, removed the supernatant, according to the mass-volume ratio of 1:8- Resuspend the precipitate in PB solution at a ratio of 10, then break up the bacteria with ultrasonic waves, wash the inclusion bodies obtained after ultrasonic treatment with 2 mol/L urea for 2 to 4 times, and then add the inclusion bodies to 8 mol/L urea, The dissolved inclusion body solution was obtained, and Sephacryl-200 (S-200) was used as a column filler, and the dissolved supernatant was subjected to gel filtration chromatography, and then the obtained purified sample was placed in a refolding buffer at 4°C for a refolding reaction for 72 hours , and finally concentrated slowly with PEG 6000 to obtain human interleukin-22 protein.

The host cell described in the above method can be E.coliDH5α, HB101 or JM109, etc.

The Hanks solution described in the above method can be purchased directly from the market, such as from Tianrun Shanda Company.

The lymphocyte separation liquid described in the above method can be purchased directly from the market, such as from Shanghai Huajing Biological Company.

The ConA described in the above method can be purchased directly in the market, such as from Chas Biotech.

The anti-CD ₃ described in the above method can be purchased directly in the market, for example, it can be purchased from Shanghai Wowu Biotechnology Co., Ltd.

The above-mentioned 1640 culture medium is a commonly used medium, which can be purchased from the market, such as from Tianrun Shanda Reagent Company.

The above-mentioned total RNA extraction kit can be purchased from the market, for example, from Promega.

The PB solution described in the above method is a mixed solution with a final concentration of 100mmol/L Na ₂ HPO ₄ and 100mmol/L NaH ₂ PO ₄ . See page 1568 of the third edition of "Molecular Cloning Experiment Guide".

The above-mentioned LB medium refers to a commonly used LB medium.

For the methods of transforming host cells by electroporation or CaCl ₂ method in step (3) and step (4) of the above method, see 96-99 of the third edition of Molecular Cloning Experiment Guide.

The gel filtration chromatography uses Sephacryl-200 (S-200) as a column filler.

The composition and ratio of the refolding buffer are: 100mmol/L Na ₂ HPO ₄ , 100mmol/L NaH ₂ PO ₄ , 0.1mmol/L GSSG, 1mmol/L GSH, 0.1% PEG.

The advantages of the present invention: (1) the present invention provides a new application of interleukin-22, which expands the scope of application of interleukin-22; (2) the present invention has a high expression level, and the target protein can occupy bacterial protein (3) The method of the present invention uses pBV220 as a carrier, adopts a temperature-induced induction mode, and has low production cost.

Description of drawings

Figure 1 is the electrophoresis diagram of human peripheral blood total RNA

Figure 2 is the electrophoresis diagram of RT-PCR products

1. DL2000

2. hIL22 cDNA

Figure 3 is the electrophoresis diagram of pBV220-IL22 digestion product

1. DL2000

2. Electropherogram of pBV220 product digested with EcoRI and BamHI

3. Electropherogram of pBV220-hIL-22 product digested with EcoRI and BamHI

Figure 4 is the electrophoresis diagram of expression products of hIL22 in various Escherichia coli

1. Marker

2. pBV220/JM109

3. pBV220-IL22/JM109

Figure 5 is the electropherogram of the purification of hIL22

Figure 6 is the elution curve of hIL22

Figure 7 is a graph showing the pro-proliferation effect of hIL22 on LO2 cells

Figure 8 is the electrophoresis diagram of c-myc expression level in LO2 cells

1. DL2000

2. Total RNA products extracted from LO2 cells

3. Effects of different concentrations (20, 200, 400, 800ng/mL) of hIL22 on the expression level of c-myc

Figure 9 Electropherogram of expression level of bcl-2 in LO2 cells

1. DL2000 DNA marker;

2. LO2 cell total RNA reverse transcription product

3-6. Expression levels of bcl-2 in LO2 cells with different concentrations of interleukin-22 (20, 200, 400, 800 ng/mL)

Figure 10 Histopathological slices of nonalcoholic fatty liver disease treated with hIL22

A: normal group; B: model group; C: IL22 treatment group

Detailed ways

Example 1

Preparation of hIL22

(1) Capture of hIL-22 gene and construction of recombinant expression vector

a. Extraction of total RNA Dilute 1ml of human fresh peripheral blood by 2 times with PBS containing 2mmol/L EDTA, spread it on an equal volume of lymphocyte separation medium (purchased from Shanghai Huajing Biological Co., Ltd.), and then run it at 800r/min Centrifuge to make layers, and then use capillaries to suck out the cells of the gray and white coat; wash the gray and white coat cells with Hanks solution (purchased from Tianrun Shanda Co., Ltd.) Cells were cultured at 37°C in a 5% CO ₂ incubator for 24 hours, and then ConA (2mg/ml, purchased from Shasi Biotechnology Co., Ltd.) and anti-CD ₃ (4mg/ml, purchased from Shanghai Wuwu Biotechnology Co., Ltd. Company) co-stimulatory cells, then at 37 ° C, 5% CO ₂ Continue to cultivate in the incubator for 24 h, then use the total RNA extraction kit (Promega company) to extract the total RNA (operation is carried out according to the instruction manual), to obtain the total RNA (see Figure 1 );

b. Isolation of target gene and construction of pBV-IL22 plasmid Take 5uL of total RNA, use f1 and r1 as primers, use Oligo(dT) for reverse transcription, and synthesize the first-strand cDNA; the primers used are:

EcoRI f1: 5′GAGAATTCATATGGCACCCATCAGCTCCCA3′

BamHI r1: 5′ACGGATCCTCAAATGCAGGCATTTCT3′

The reverse transcription system is as follows: total RNA 5uL, RNase inhibitor 0.5uL, Oligo(dT) 0.5uL, DEPC-treated water 6.5uL, a total of 12uL, 70°C for 5min to 4°C;

RT: Add to the above system: 5×buffer 4uL, 2.5mMdNTP 2uL, AMV 1uL, RNase inhibitor 1uL, 42℃ for 1h, 85℃ for 5min to 4℃;

Use the first-strand cDNA generated by reverse transcription as a template for PCR reaction. PCR reaction system: 10PC reaction × buffer 2.0uL, 2.0mmol/L dNTP 2.0uL, 5pmol/uL f1 2.0uL, 5pmol/uLr1 2.0uL, reverse Transcript product 4.0uL, TaqDNA polymerase 0.5uL, ddH ₂ O 7.5uL, total 20uL;

PCR reaction conditions are 94°C pre-denaturation for 3min; 94°C for 30S, 50°C for 30S, 72°C for 30S, 30 cycles; 72°C for 7min; use 1.2% agarose gel to separate the PCR product, recover the PCR product, and then use EcoRI Digested with BamHI, then ligated with pBV220 double cut by the same enzyme with ligase, transformed E.coliDH5α competent cells with the ligated product, and sent the recombinant plasmid identified as positive by double digestion to BioaSia company for sequencing, the result ( See Fig. 2 and Fig. 3) obtain the recombinant plasmid with target gene hIL22, this plasmid is marked as pBV-IL22.

(2) Expression of pBV-IL22 in Escherichia coli The constructed plasmid pBV-IL22 was transformed into Escherichia coli competent cell JM109, and monoclonal colonies were picked from the transformation plate and inoculated in 3ml amp ^r LB medium (LB medium: Weigh 10 g of tryptone and 5 g of yeast extract in the liquid medium, dilute to 1 L with distilled water, and activate overnight at 30°C. The next day, inoculate the activated bacteria in 3ml of fresh amp ^r LB medium at a ratio of 3%, and shake at 30°C until the logarithmic growth phase until the OD _600nm reaches 0.5, then transfer the bacteria directly to a water bath shaker at 42°C, 200rpm Centrifuge at 4°C and 12,000rpm for 10 minutes to collect the bacteria, and resuspend the pellet in PB (100mmol/L Na ₂ HPO ₄ , 100mmol/L NaH ₂ PO ₄ ) at a mass volume ratio of 1:10 , the cells were disrupted by ultrasonic waves (6 times, 30s each time; +200W), the supernatant and the precipitate were collected respectively, and 15% SDS-PAGE was performed. The results (see Figure 4) showed that hIL22 was expressed in E.coli JM109.

(3) The crushed precipitate of the purified bacteria of rhIL-22 was washed with 10 times the volume of 2 mol/L urea, and the washed inclusion bodies were dissolved in 8 mol/L urea, left at room temperature for 8 hours, and kept stirring until the inclusion bodies were fully Dissolve, then centrifuge at 4°C and 12000rpm for 30min, collect supernatant and precipitate respectively, select Sephacryl-300 (S-300) as filler, column volume is 3.6×100cm, and separate and purify the target protein. The column was equilibrated with 2 times the volume of equilibrium solution (8mol/L urea, 100mmol/L Na ₂ HPO ₄ , 100mmol/L NaH ₂ PO ₄ , 50mmol/LNaCl PH7.4, 10mmo/L DTT) before loading the sample. About 2% of the column volume, use the eluent (50mmol/L Na ₂ HPO ₄ , 50mmol/L NaH ₂ PO ₄ , 50mmol/L NaCl) to elute the sample at a flow rate of 0.5mL/min; use 15% SDS-PAGE The purity of the sample corresponding to each elution peak was analyzed, and as a result (see Figure 5) the purified rhIL-22 protein was obtained;

(4) Refolding of rhIL-22 Add refolding buffer (50mmol/L Na ₂ HPO ₄ , 50mmol/L NaH ₂ PO ₄ , 0.1mmol/L GSSG, 1mmol/L GSSG to the purified rhIL-22 protein obtained in (3). LGSH, 0.1% PEG), the protein concentration was controlled at 0.15 mg/ml, placed at 4°C for 50 hours, combined with dialysis to gradually reduce the urea concentration, and finally concentrated slowly with PEG6000 to obtain rhIL-22 protein.

Example 2

Proliferation-promoting activity of hIL22 on LO2 cells

Culture LO2 cells with RPMI1640 culture medium containing 10% fetal bovine serum (purchased from Gibco BRL Company) until the cells grow well, collect the cells, and inoculate them in 96-well cell culture plates at 1× ¹⁰⁵ cells/mL, 50 μl /well; then add 50 μl of the hIL-22 protein obtained in the doubling dilution embodiment 1 to each well, and the final concentrations are respectively 0.1, 1, 2.5, 10, 50, 100, 1000, 2000, 4000 and 8000ng/ml, At the same time, set a blank control without cells and a negative control with cells but no factors added, and do three repetitions for each concentration; after culturing in a 5% CO ₂ incubator at 37°C for 72 hours, add 20 μl of 5 mg/ml MTT to each well Continue culturing for 5 hours, then add 100 μl of 10% SDS (dissolved in 0.01N HCl) to each well, and measure the absorbance at 570 nm after the purple formazan crystals are dissolved.

The concentration of hIL-22 after renaturation and concentration was measured at 1 mg/mL by Bradford method, and the promotion of the proliferative activity of LO2 cells by the recombinant protein was detected by MTT method. The results (see Figure 7) compared with the negative control, after the recombinant protein hIL-22 was added, the proliferation of LO2 cells was significantly changed; with the increase of the concentration of recombinant protein, the proliferation activity of hIL-22 to promote the proliferation of LO2 cells was significantly strengthened, at 1 μg At 4 μg/mL, the recombinant protein hIL-22 had the highest proliferative activity in promoting the proliferation of LO2 cells, and then the activity began to decline. After reaching 4 μg/mL, the proliferation of LO2 cells appeared a plateau effect. The measured values were statistically tested, and the results showed that the absorbance at 570nm of the LO2 cells stimulated by different concentrations of the recombinant protein in the experimental group was significantly different from that of the negative control (P<0.01), indicating that hIL-22 promoted the proliferation activity of the LO2 cells .

Example 3

Effect of hIL22 on the expression level of c-myc in LO2 cells

LO2 cells were cultured in a 60 mm culture dish for 2 h, and then grouped into groups and sequentially added the recombinant IL-22 protein solution prepared in Example 1 so that the final concentrations of various proteins were 20, 200, 400, and 800 ng/mL, respectively. After culturing at 37°C and 5% CO ₂ for 2 hours, RNA was extracted and quantitatively carried out by RT-PCR. The results (see Figure 8) showed that after being stimulated by exogenous hIL-22, the expression level of c-myc gene gradually increased from 20 to 800 ng/mL, which was dependent on the concentration of hIL-22, indicating that hIL22 can increase the c - expression level of myc.

Example 4

Effect of hIL-22 on the expression level of bcl-2 in LO2 cells

After acting on LO2 cells with hIL-22 recombinant protein solutions obtained in Example 1 at different concentrations, the final concentrations of various proteins were respectively 20, 200, 400, and 800 ng/mL, and RNA was extracted and reverse-transcribed quantitatively to obtain Specific bcl-2 primers were used for PCR, and the amplified products were identified by 1% agarose gel electrophoresis. It can be seen that the sizes of bcl-2 and hGAPDH-specific target bands were basically consistent with the expected 435bp and 445bp respectively, stimulated by exogenous hIL-22 Finally, the results showed (see Figure 9) that the expression level of bcl-2 gene gradually increased from 20 to 800 ng/mL, which was dependent on the concentration of hIL-22, indicating that hIL-22 promoted the expression of bcl-2 in LO2 cells.

Example 5

Effect of hIL22 on Alcoholic Liver Disease

(1) Establishment of alcoholic liver disease model Select 60 male Wistar rats of 200-250 g (purchased from the Experimental Animal Center of the Academy of Military Medical Sciences), 10 in the normal group and 50 in the model group. With 56° Red Star Erguotou (purchased from Beijing Red Star Co., Ltd.), the dosage is:

1 to 4 weeks 16ml/kg.d, twice a day

5-8 weeks 24ml/kg.d, twice a day

9-12 weeks 32ml/kg.d, twice a day

The high-fat feed consisted of basal feed + 10% lard oil + 5% corn oil + 1% cholesterol; the normal group was fed with full-price nutritious pellet feed, and at the same time fed with the same dose of normal saline as the model group;

(2) At the end of the 12th, the model group was divided into the experimental group and the experimental control group, and continued to be fed with a high-fat diet. The experimental group was injected with hIL22 recombinant protein through the tail vein at a dose of 0.25ug/g.d for 14 days, and the experimental group was injected continuously for 14 days. Inject the same dose of normal saline through the tail vein, anesthetize with 5% sodium pentobarbital solution 14 days later, take blood from the inferior vena cava to test related indicators, separate the liver, weigh it, and calculate the change of the liver index (liver mass/body weight). Liver tissue was taken, fixed in 10% formaldehyde, paraffin sectioned, stained with HE, and its pathological score was evaluated. The results (see Table 1) showed that IL22 can significantly reduce the increase of serum transaminases and effectively alleviate the pathological changes of alcoholic liver disease.

Example 6

Effect of hIL22 on non-alcoholic fatty liver disease

(1) Establishment of non-alcoholic fatty liver disease model 30 newly weaned SD rats (purchased from the Experimental Animal Center of the Academy of Military Medical Sciences) were divided into a blank group, an experimental control group, an experimental group, and the blank group was fed with full-price Nutritious pellet feed, the experimental group and the control group were fed with high-fat feed, the composition of high-fat feed was 70% basic feed + 20% lard oil + 2% cholesterol + 1% bile salt + 7% egg yolk powder, the modeling cycle for 12 weeks;

(2) At the end of the 12th, the experimental group and its blank group were fed with high-fat feed, while the experimental group was injected with recombinant hIL22 protein (0.25ug/g.d), and the control group was injected with the same dose of normal saline; the treatment cycle was 14 days, After 4 days, anesthetized with 5% pentobarbital sodium solution, blood was collected from the inferior vena cava to test related indicators, the liver was separated, weighed, and the change of liver index (liver mass/body weight) was calculated, liver tissue was taken, fixed with 10% formaldehyde, Paraffin sections, HE staining, and evaluation of the pathological score, the results (see Table 2, Figure 10) show that hIL22 can significantly reduce the increase in serum enzymes and blood lipids, improve liver function, and reduce the pathological score of non-alcoholic fatty liver disease .

Table 1: The therapeutic effect of hIL22 on alcoholic liver disease

Number of cases weight(g) liver index ALT(U/L) AST(U/L) r-GT(U/L) TC(mg/g) TG(mg/g) normal group 8 334.4±25.2 2.5±0.12 44.8±4.3 71.4±10.8 21.5±5.8 61.3±6.3 34.5±2.2 model group 8 263.8±67.4 4.4±0.7 93.9±6.9 79.1±6.5 81.4±11.3 77.5±5.7 93.9±6.9 therapy group 8 288.1±39.7 3.8±0.2 72.3±7.7 69.0±6.1 63.4±8.7 68.7±6.3 72.3±7.7

Table 2: The therapeutic effect of hIL22 on non-alcoholic fatty liver disease

Number of cases weight(g) liver index ALT(U/L) AST(U/L) MDA(ug/g) TG(mg/g) TC(mg/g) normal group 8 334.4±25.2 2.5±0.1 51.7±7.9 80.3±13.5 5.6±0.32 53.2±6.3 55.2±4.7 model group 8 560.8±20.7 5.4±0.3 132.8±22.5 156.7±22.5 8.9±0.31  163.2±33.4 89.4±6.3 therapy group 8 5 10.2±25.3 5.0±0.5 98.7±15.6 124.3±15.2 6.7±0.24 115.9±18.4 68.7±6.3

Claims (9)

1. the application of interleukin-22 in preparation treatment liver disease drug;

2. according to the described application of claim 1, it is characterized in that described interleukin-22 is meant a kind of of human interleukin-2 2, recombination human interleukin-22, Mus interleukin-22 or reorganization Mus interleukin-22 etc.

3. according to claim 1 or 2 described application, it is characterized in that described hepatopathy is meant a kind of of hepatopathy such as depletion after alcoholic liver disease, non-alcohol fatty liver, viral hepatitis, acute hepatic failure, hepatic fibrosis, hepatic ischemia-reperfusion injury, the liver transplantation.

4. according to the described application of claim 3, it is characterized in that described hepatopathy is meant alcoholic liver disease or non-alcohol fatty liver.

5. according to the described application of claim 4, it is characterized in that described hepatopathy is meant alcoholic liver disease.

6. according to the described application of claim 5, it is characterized in that described hepatopathy is meant non-alcohol fatty liver.

7. the proteic preparation method of claim 2 described recombination human interleukin-22 comprises the steps:

(1) utilizes the fresh peripheral blood of people, cultivate human peripheral lymphocyte, extract total RNA;

(2) being template with total RNA, is that primer carries out the RT-PCR amplification with f1 and r1, gets the cDNA sequence of hIL22; The sequence of described primer f1 and r1 is as follows:

f1：5′GAGAATTCATATGGCACCCATCAGCTCCCA3′；

r1：5′ACGGATCCTCAAATGCAGGCATTTCT 3′

And then be template with the cDNA of gained, be that primer carries out pcr amplification with f1 and r1, the DNA sequence of hIL22;

(3) with EcoRI and BamHI respectively with DNA sequence and the plasmid pBV220 enzyme action of step (2) gained hIL22; Then both are mixed, both are connected, will connect the product electricity consumption then and transform or CaCl with the T4 ligase ₂The conversion method transformed host cell, Screening and Identification gets recombiant plasmid pBV220/hIL22;

(4) electricity consumption transforms or CaCl ₂Method is with recombiant plasmid pBV220/hIL22 transformed host cell; Be inoculated on the LB culture medium and cultivate, centrifugal then, resuspended precipitate utilizes the ultrasonic disruption thalline, then with the degeneration of gained inclusion body, dissolving, purification, renaturation, concentrated, promptly gets recombination human interleukin 22 albumen of the present invention.

8. in accordance with the method for claim 7, it is characterized in that its detailed operating procedure is:

(1) with the fresh peripheral blood of people with 2 times of the PBS solution dilutions that contains 2mmol/L EDTA, be laid on then on the equal-volume lymphocyte separation medium; Centrifugal under 700～1000rpm again, use the greyish white confluent monolayer cells of glass capillary sucking-off then, wash greyish white confluent monolayer cells 2～3 times with Hanks liquid, more greyish white confluent monolayer cells is suspended in 1640 and cultivates in the base, and at 37 ℃, 5%CO ₂Cultivate 24hr in the incubator, then add ConA and anti-CD ₃Be total to irritation cell, again at 37 ℃, 5%CO ₂Continue in the incubator to cultivate 24h, extract total RNA with the total RNA extraction reagent box;

(2) being template with total RNA, is primer with f1 and r1

f1：5′GAGAATTCATATGGCACCCATCAGCTCCCA3′

r1：5′ACGGATCCTCAAATGCAGGCATTTCT 3′

Carry out RT-PCR amplification and obtain the cDNA sequence of human interleukin-2 2;

The each several part ratio of described RT-PCR reaction system is: total RNA, 5ul; The RNase inhibitor; 0.5ul; Oligo (dT), 0.5ul; The DEPC treating water, 6.5ul; Amount to 1 2ul; 70 ℃ 5min to 4 ℃; RT: add in above-mentioned system: 5 * buffer 4ul, 2.5mMdNTP 2ul, AMV 1ul, RNase inhibitor 1ul, 42 ℃ of 1h, 85 ℃ 5min to 4 ℃;

The first chain cDNA that produces with reverse transcription is a template then, with f1, r1 is that primer carries out the PCR reaction, the each several part ratio of described PCR reaction system is: 10PCR reaction * buffer 2.0ul, 2.0mmol/LdNTP 2.0ul, 5pmol/ul f1 2.0ul, 5pmol/ul r1 2.0ul, reverse transcription product 4.0ul, TaqDNA polymerase 0.5ul, ddH ₂O 7.5ul, 20ul altogether; Described PCR reaction condition is 94 ℃ of pre-degeneration 3min, 94 ℃ of 30S, 50 ℃ of 30S, 72 ℃ of 30S, 30 circulations; 72 ℃ are extended 7min; Agarose gel with 1.2% separates the PCR product;

(3) add restricted enzyme EcoRI and BamHI to RT-PCR product and plasmid pBV220 respectively, enzyme action 3.5h in 37 ℃ of water-baths presses 10～4: 1 mixed with both then, adds the T4 ligase, carry out coupled reaction 12h, conversion of utilization electricity or CaCl in 16 ℃ ₂Method will connect the product transformed host cell, utilize amicillin resistance screening recombinant clone, and extract phase answers plasmid, enzyme action to identify, check order, and gets recombiant plasmid pBV220/hIL22;

(4) electricity consumption transforms or CaCl ₂Method is transformed into recombiant plasmid pBV220/hIL22 in the host cell; Transform bacterium colony and spend the night for 30 ℃, be inoculated in LB culture medium according to 1～2% percent by volume next day, cultivates 2～3hr at 30 ℃, places then under 42 ℃ of shaking baths, the 180～220rpm condition and cultivate 4～8hr; Again at 4 ℃, 10,000～12, centrifugal 10～15min under the 000rpm condition, remove supernatant, ratio according to mass volume ratio 1: 8～10 is resuspended in precipitation in the PB solution, again with the ultrasonic disruption thalline, the inclusion body washing that obtains after with ultrasonic Treatment with 2mol/L carbamide 2～4 times, inclusion body is joined in the 8mol/L carbamide then, getting dissolved inclusion body solution, is column packing with Sephacryl-200 (S-200), and the dissolving supernatant is carried out gel permeation chromatography, again the gained purification of samples is placed 4 ℃ of renaturation buffers to carry out renaturation reaction 72hr, slowly concentrate with PEG 6000 at last and obtain human interleukin-2 2 albumen.

9. according to claim 7 or 8 described methods, it is characterized in that described host cell is E.coliDH5 α, HB101 or JM109.

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