CN102775488B - Colorectal cancer specific antigen peptide and colorectal cancer detection kit - Google Patents

Abstract

The invention discloses a colorectal cancer-specific antigen peptide, an expression carrier and a preparation method of the colorectal cancer-specific antigen peptide, a colorectal cancer-specific autoantibody ELISA kit containing the expression carrier and an operation method thereof. Use phage to display the colorectal cancer cDNA library on the surface of the phage, and screen the expression vector of the colorectal cancer-specific antigen peptide through affinity screening and serological analysis. And stop solution, etc. constitute the detection kit. The expression carrier of the colorectal cancer-specific antigen peptide of the present invention effectively expresses the colorectal cancer-specific antigen peptide, is coated on a microtiter plate, and has the ability to detect autoantibodies produced by tumor stimulation in the serum of colorectal cancer patients, thereby achieving The purpose of early detection and screening of colorectal cancer patients.

Description

Colorectal cancer specific antigen peptide and colorectal cancer detection kit

This application is a divisional application of the Chinese patent application number 201010275415.1, the application date is September 7, 2010, and the invention title is "colorectal cancer specific antigen peptide and colorectal cancer detection kit".

technical field

The invention relates to the technical field of medical detection reagents, and relates to an expression vector and a construction method of a colorectal cancer-specific antigen peptide, a colorectal cancer-specific autoantibody ELISA detection kit and an operation method.

Background technique

Colorectal cancer is one of the common malignant tumors, and its incidence rate ranks third in the world. In my country, it has a rising trend year by year, and in Shanghai, it ranks second in the incidence rate of malignant tumors. According to the 2007 statistical data of the Ministry of Health of China, the incidence of cancer ranks third among all malignant tumors, and the mortality rate of colorectal cancer is 10.25 per 100,000, ranking fifth among cancer deaths. Colorectal cancer can be divided into stages Ⅰ, Ⅱ, Ⅲ, and Ⅳ according to TNM staging. The prognosis of each stage of colorectal cancer is very different. Early colorectal cancer has a low recurrence and metastasis rate and a good prognosis. The 5-year survival rate after surgery can reach 97%. The 5-year survival rate after surgery for advanced colorectal cancer is only 40%-50%. Therefore, it is very important to explore early diagnosis and screening methods for colorectal cancer.

Currently, the commonly used detection methods for colorectal cancer include digital rectal examination, fecal occult blood test, colonoscopy and imaging examination. However, these examinations have shortcomings, such as the limited range of digital rectal examination, poor sensitivity and specificity of fecal occult blood test, colonoscopy is painful and difficult for patients to accept, and imaging examination is not sensitive to early tumors. Currently commonly used tumor markers such as carcinoembryonic antigen (CEA), CA19-9 and other indicators lack sensitivity and specificity, and cannot be used for early diagnosis of colorectal cancer. Therefore, it is urgent to establish a specific detection method for high-throughput diagnosis of colorectal cancer.

Contents of the invention

After the tumor occurs, the characteristic antigens on the surface of tumor cells or the characteristic antigens secreted by tumor cells stimulate the body to produce antibodies, and these antibodies can exist in the serum of asymptomatic early-stage tumor patients. By looking for the characteristic antigen of colorectal cancer and using it to detect the specific autoantibody in the patient's serum as a molecular marker, colorectal cancer can be detected early. Compared with other current methods, it is minimally invasive, simple, economical and sensitive And high specificity, it is convenient to be used in the screening of community population, so as to achieve the purpose of early detection of colorectal cancer.

The first object of the present invention is to provide a colorectal cancer-specific antigen peptide, which can be used to detect specific autoantibodies in patient serum, so as to use it as a molecular marker for early detection of colorectal cancer.

The second object of the present invention is to provide an expression vector of colorectal cancer specific antigen peptide.

The third object of the present invention is to provide a method for constructing the expression vector.

The fourth object of the present invention is to provide a colorectal cancer-specific autoantibody ELISA kit comprising an expression vector for detecting early colorectal cancer.

The fifth object of the present invention is to provide the preparation and operation method of the kit.

The colorectal cancer-specific antigen peptide provided by the present invention has an amino acid sequence as shown in SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, or SEQ ID No.10.

The expression vector of the colorectal cancer-specific antigen peptide provided by the present invention is composed of a phage and a cDNA fragment encoding a colorectal cancer-specific antigen peptide, and the sequences of the cDNA fragment are as SEQ ID No.1, SEQ ID No.3, and SEQ ID No. .5, as shown in SEQ ID No.7 or SEQ ID No.9. Preferably, the phage is T7 phage.

The method for constructing a colorectal cancer-specific antigen peptide expression vector provided by the present invention includes:

a) Construction of colon cancer phage display peptide library:

Total RNA was extracted from 30 cases of fresh colorectal cancer tissues, mixed in equal amounts to 1 mg, and then mRNA was extracted, reverse-transcribed into cDNA with oligodT primers, the ends of the cDNA were blunted, EcolⅠ and Hind III adapters were added, and small enzymes were removed after double enzyme digestion. The fragments were then inserted into the arms of T7 phage for in vitro packaging to construct a colon cancer phage display peptide library;

b) Affinity screening of colorectal cancer-specific antigen peptides:

Take 10 μl of A/G agarose beads and place them in a 1.5ml centrifuge tube, wash them twice with 500 μl of PBS at pH 7.4, block with 1% BSA for 1 hour at 4°C, and mix the agarose beads with 15 μl of plasma from colorectal cancer patients and control patients respectively. Incubate overnight at ℃, wash 3 times with 500 μl PBS, dissolve the enriched antibody with 10 μl PBS, combine 10 tubes of colorectal cancer antibody and 10 non-colorectal cancer antibody into one tube, and take the phage display peptide library amplification solution 20 μl, first incubated with 20 μl of non-colorectal cancer antibody for 1 hour, and the unbound supernatant was combined with 20 μl of colorectal cancer antibody, the phage bound to the agarose beads were retained, and eluted with 100 μl of 1% SDS, and then transfected into bacteria Increase, complete a round of affinity screening, repeat the above steps, and repeat five rounds of affinity screening;

c) Serological screening of molecular markers for early detection of colorectal cancer:

c1) Primary screening of mixed blood samples:

After the screened phage display peptide library was diluted with LB liquid medium 1: ¹⁰⁸ , take 100 μl of phage liquid, 250 μl of freshly shaken BLT5403 bacteria, 3 ml of top layer agar kept at 55°C, mix well and immediately pour into LB After cooling, incubate in a 37°C incubator for 4 hours, and a single phage plaque can be seen. Add 1ml of freshly shaken bacteria BLT5403 to each 1.5ml centrifuge tube, and pick a single one with a clear border at random. A total of 2,000 phage clones were randomly selected in a 1.5ml centrifuge tube and numbered sequentially. The selected phages were placed in a constant temperature shaker at 37°C for more than 4 hours until the liquid in each tube became clear. Store good phage clones at 4°C, use T7 primers for PCR reaction, amplify the insert fragments of the selected phage clones, and pick phage clones with amplified fragments of more than 200 bp for further screening by ELISA;

The steps of the ELISA experiment are:

Dilute the T7TAILER antibody with PBS of pH 7.4 at a ratio of 1:1000, take 100 μl from each well and coat it on a 96-well microtiter plate, shake gently overnight at 4°C;

Wash the plate with washing solution, 300 μl per well, wash four times, about 1 minute each time, pat dry; use 200 μl 2% BSA/PBS to block at 26°C for 2 hours;

Wash the plate with washing solution, 300 μl per well, wash four times, about 1 minute each time, pat dry; add 100 μl of phage diluted 1:5 with 1% BSA, add 4 wells for each sample; add Empty phages without insert fragments were used as negative control 4 wells and blank control 2 wells, incubated at room temperature for 2 hours, discarded the liquid in the wells, and patted dry;

For each phage sample, add 100 μl of 1% BSA diluted 1:500 mixed plasma of the colorectal cancer group and 2 wells of the mixed plasma of the control group. The patient information of the mixed plasma of the colorectal cancer group and control group is the same as that used in step b) for affinity screening. For patients, incubate at room temperature for 1 hour;

After washing the plate, add 100 μl 1:10000 diluted HRP-coupled goat anti-human IgG to each well, and incubate at 26°C for 1 hour;

After washing the plate, add 2 drops of chromogenic reagents A and B each at room temperature, mix well, and incubate at 37°C for 5 minutes, add 25 μl of stop solution, and immediately use a microplate reader to detect with a wavelength of 450 nm. Set the well to zero, then read the OD value of each well, and record the result. Each time the ELISA is performed, the following formula is used to calculate and judge the result: the average OD value of the sample/the average OD value of the negative control, the ratio ≥ 2.0, it is judged as a positive result ; The average OD value of the sample/the average OD value of the negative control, if the ratio is <2.0, it is judged as a negative result; find out the meaningful phage clones that are positive in the mixed serum reaction with colorectal cancer, but negative in the mixed serum reaction with the control;

c2) Independent serum re-screening:

The meaningful phages after the primary screening in step c1) were re-screened by ELISA with the sera of 30 cases of colorectal cancer patients and 30 cases of healthy controls. The steps were the same as those of the ELISA experiment described in c1, except that The serum is independent, rather than mixed serum, compared the reactivity of the meaningful phage with the sera of patients with large intestine cancer and the control serum, and used the method of independent sample t test to select 30 cases of colorectal cancer serum and 30 cases of colorectal cancer. Phage clones with significantly different response data from healthy control sera;

c3) Acquisition of diagnostic module of colorectal cancer-specific antigen peptide recombinant vector:

All independent serum samples were randomly sampled 2,000 times using the random forest method using dige R software, and the screening ability of the phages with significant differences obtained in step c2 was sorted from high to low according to the ability of each phage peptide to distinguish colorectal cancer serum ;Adopt the Bayesian bivariate model to test the effect of the joint detection of phages in turn, and the sensitivity and specificity of the combined detection of phages with significant differences obtained in step c2 are the closest according to the least number of joints and the sensitivity and specificity of joint detections According to the standard, the expression vectors of 5 colorectal cancer-specific antigen peptides were obtained, and the sequences of cDNA fragments inserted on them were as SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID Shown in No.9.

The colorectal cancer-specific autoantibody ELISA kit provided by the present invention includes T7TAILER monoclonal antibody, ELISA plate, negative control, enzyme conjugate, chromogenic reagent A&B, and stop solution, wherein the kit also includes The expression vectors of 5 colorectal cancer-specific antigen peptides, the expression vectors of the 5 colorectal cancer-specific antigen peptides are all composed of T7 phage and cDNA fragments encoding colorectal cancer-specific antigen peptides, the sequences of the cDNA fragments are as follows: Shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, and the negative control is empty phage, and the enzyme conjugate is HRP-coupled Goat anti-human IgG.

The operation method of the colorectal cancer-specific autoantibody ELISA kit provided by the present invention includes:

A) Dilute T7TAILER antibody at 1:1000 dilution with 1×PBS with pH 7.4, 100ul per well, coat on 96-well ELISA plate, shake gently overnight at 4°C; use 1× washing solution, 300ul per well, wash four times, about 1 minute each time, pat dry and seal each well with 2%BSA/PBS 200ul at 26°C for 2 hours; 1× washing solution, 300ul per well, wash four times, about 1 minute each time Minutes, pat dry, add 100ul of 5 recombinant vectors expressing colorectal cancer-specific antigen peptide diluted 1:5 in 1%BSA, add 2 wells for each specimen; add empty phage without insert fragments in each test as negative control and blank For each 2 wells of the control, incubate at room temperature for 2 hours, discard the liquid in the wells, and pat dry;

B) Add 100ul1%BSA1:500 diluted colorectal cancer patient plasma to each well, incubate at room temperature for 1 hour, wash the plate with 300ul per well, wash four times, about 1 minute each time, and pat dry; add 100ul1:10000 to each well Diluted HRP-coupled goat anti-human IgG, incubate at room temperature for 1 hour, wash the plate with washing solution, 300ul per well, wash four times, about 1 minute each time, pat dry; add chromogenic reagent A at room temperature to each well 2 drops each of B and B, mix well, incubate at 37°C for 5 minutes, add 25ul of stop solution, immediately detect with a microplate reader, take a wavelength of 450nm, first use a blank well to zero, then read the OD value of each well, and record result;

C) Calculate and judge the result according to the following formula: after zeroing the blank hole, the average OD value of the sample/the average OD value of the negative control, substitute the result into the joint screening model, set the cut-off value to 0.5, and judge when the result is ≥0.5 positive; if the result is <0.5, the result is judged as negative, wherein, the joint screening model is obtained by summarizing 5 recombinant vectors expressing colorectal cancer-specific antigen peptides, serum from 30 patients with colorectal cancer and 30 healthy controls Based on the serum response data, a Bayesian bivariate regression model for screening colorectal cancer was established using Binary Regression Models Version 2.0 software in the Matlab 7.0 environment.

The present invention uses T7 phage to display the colorectal cancer cDNA library on the surface of the phage, and screens out the expression vector of the colorectal cancer-specific antigen peptide through affinity screening and serological analysis, and the colorectal cancer-specific autoantibody ELISA containing the expression vector The detection kit has the ability to detect autoantibodies produced by tumor stimulation in the serum of patients with colorectal cancer. Combining multiple screened antigen peptides can achieve the purpose of early detection and screening of colorectal cancer patients.

Description of drawings

Figure 1 is an optical picture of the original library of the colon cancer phage display peptide library.

Figure 2 is a PCR amplification diagram of randomly selected plaques.

Fig. 3 is a schematic diagram of a mathematical model constructed based on the results of joint screening of 30 pairs of training group sera by 16 phages.

Figure 4 (a) and (b) are the ROC curves for the joint screening of 16 phage clones and the joint screening of 5 phages, respectively.

Fig. 5 is a schematic diagram of a mathematical model constructed based on the results of joint screening of 30 pairs of training group sera by 5 phages.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following examples are only used to illustrate the present invention but not to limit the scope of the present invention.

Example 1 Construction of colon cancer phage display peptide library

Extract total RNA from 30 cases of fresh colorectal cancer tissues, mix equal amounts to 1 mg, extract mRNA, use oligodT primers to reverse transcribe into cDNA, blunt the ends of cDNA, add EcolⅠ and Hind III adapters, and remove the small RNA after double enzyme digestion. The fragments were then inserted into the arms of T7 phage (NOVAGEN), packaged in vitro, and constructed into a colon cancer phage display peptide library, and its optical picture is shown in Figure 1. The capacity of the constructed phage display peptide library was determined to be 3.0×10 ⁶ pfu, and the recombination rate was 60%, which met the requirements of the screening test.

Example 2 Affinity Screening of Colorectal Cancer Specific Antigen Peptides

Take 10 μl A/G agarose beads and place them in a 1.5ml centrifuge tube, wash twice with 500 μl PBS (pH7.4), and block with 1% BSA at 4°C for 1 hour. The agarose beads were incubated overnight at 4°C with 15 μl of serum from colorectal cancer patients and control patients, where the control patients were those who excluded any malignant tumors and precancerous lesions, and those who excluded adenomatous polyposis and hereditary nonpolyposis colorectal cancer. After washing 3 times with 500 μl of PBS, dissolve the enriched antibody with 10 μl of PBS. 10 tubes of antibodies against colorectal cancer and 10 tubes of antibodies against non-colorectal cancer were combined into one tube. Take 20 μl of the phage library amplification solution, first incubate with 20 μl of non-colorectal cancer antibody for 1 h, then combine the unbound supernatant with 20 μl of colorectal cancer antibody, keep the phage bound to the agarose beads, and wash it with 100 μl of 1% SDS, It transfects bacteria to amplification and completes a round of affinity selection. In order to enrich specific display peptides that can bind to colorectal cancer autoantibodies, the above steps were repeated, and five rounds of affinity screening were repeated.

Example 3 Serological screening of molecular markers for early detection of colorectal cancer

3.1 Preliminary screening of mixed blood samples

After the screened peptide library was diluted with LB liquid medium 1: ¹⁰⁸ , take 100μl phage liquid, 250μl freshly shaken BLT5403 bacteria, 3ml top layer agar warmed at 55°C, mix well and immediately pour it into a plate covered with LB After cooling, incubating in a 37°C incubator for 4 hours, it can be seen that a single phage plaque is formed. Add 1ml of freshly shaken bacteria BLT5403 to each 1.5ml centrifuge tube, use a sterilized toothpick to randomly pick a single, well-defined phage in a 1.5ml centrifuge tube, randomly select 2000 phage clones, and Numbered sequentially. The selected phages were shaken in a constant temperature shaker at 37°C for more than 4 hours until the liquid in each tube became clear. The selected phages were stored at 4°C. Use T7 primer (synthesized by Shanghai Sangon Biological Co., Ltd.) to perform PCR reaction to amplify the insert fragment of the selected phage, and the product is subjected to agarose gel electrophoresis, as shown in Figure 2, and the phage with an amplified fragment above 200BP is selected for ELISA The experiment was further screened, wherein the sequence of the T7 primer was:

Up: 5'-GGAGCTGTCGTATTCCAGTC-3'

Down: 5'-AACCCCTCAAAGACCCGTTTA-3'

Dilute the T7TAILER antibody (NOVAGEN) with PBS (pH7.4) at 1:1000, coat 100 μl per well on a 96-well ELISA plate, and shake gently overnight at 4°C on a shaker. Wash the plate with washing solution (Shanghai Kehua Biotechnology Co., Ltd.), 300 μl per well, wash four times, about 1 minute each time, and pat dry. Block with 200μl 2%BSA/PBS at room temperature (26°C) for 2 hours. Wash the plate as before, add 100 μl of phage diluted 1:5 with 1% BSA, add 4 wells for each sample. In each experiment, No. 50 empty phage without insert was added as negative control 4 wells and blank control 2 wells. Incubate at room temperature for 2 hours. The liquid in the wells was discarded and patted dry. Add 100 μl of 1% BSA diluted 1:500 to each phage sample to 2 wells of the mixed plasma of the colorectal cancer group and the mixed plasma of the control group. The patient information of the mixed plasma of the colorectal cancer group and the control group is the same as that used in the affinity screening of Example 2. That is to say, a certain phage to be tested is coated with six wells, among which two wells are added with mixed serum of patients with colon cancer, two wells are added with control mixed serum, two wells are not added with any serum as blank control, and four wells are coated with empty phage at the same time. Add the mixed serum of lung cancer patients and the mixed serum of control to two wells, respectively, as the corresponding negative controls of the first four wells. Incubate for 1 hour at room temperature. After washing the plate, add 100 μl 1:10000 diluted HRP-coupled goat anti-human IgG to each well, and incubate at room temperature at 26°C for 1 hour. After washing the plate, add 2 drops each of chromogenic reagents A and B (Shanghai Kehua Biotechnology Co., Ltd.) whose temperature has been balanced to room temperature, mix well, incubate at 37°C for 5 minutes, and add 25 μl of stop solution.

Immediately detect with a microplate reader, take a wavelength of 450nm, first use a blank well to zero, then read the optical density value (OD value) of each well, and record the results. Each ELISA is calculated and judged according to the following formula: after zeroing with a blank hole, the average OD value of the sample/average OD value of the negative control, if the ratio is ≥ 2.0, it is judged as a positive result; the average OD value of the sample/average OD value of the negative control, A ratio <2.0 was judged as a negative result. Find out the phages that are positive in the mixed serum reaction with colorectal cancer, but negative in the mixed serum reaction with the control, and carry out follow-up research as clones with screening significance. A total of 21 meaningful clones were screened out, all of which were identified by PCR as having inserted fragments Phage. The antigens displayed on the surface of these phage clones may have the potential of diagnosing colorectal cancer. They will be used to screen the sera of colorectal cancer patients and controls by ELISA test to determine the feasibility of detecting early colorectal cancer in clinical practice.

3.2 Independent serum re-screening

The 21 meaningful phages after primary screening were re-screened by ELISA with the sera of 30 cases of colorectal cancer patients and 30 cases of healthy controls. serum. Comparing the reactivity of 21 phage clones in the sera of colorectal cancer patients and control sera, using the independent sample t test, it was found that 16 phage clones were significantly different from the reaction data of 30 cases of colorectal cancer sera and 30 healthy control sera (p< 0.05).

Example 4 Application of training group serum to establish detection model

4.1 Establish a joint detection mathematical model

In order to improve the sensitivity of the detection kit, a joint detection method of different phage clones was adopted. Summarize the response data of 16 different phage clones and 30 pairs of training groups (30 cancer patients and 30 controls), and use the Binary Regression Models Version 2.0 software in the Matlab 7.0 environment to establish a Bayesian bivariate for screening colorectal cancer Mathematical model (Bayesian binary regression), which detects the probability of the tester suffering from colorectal cancer. 30 cancer patients belong to category 1, 30 controls belong to category 0, and the cut-off value is 0.5. If it is greater than or equal to 0.5, it is positive, and if it is less than 0.5, it is negative. As shown in Figure 3, this model can be used to successfully distinguish 30 pairs of training groups cancer patients and healthy controls.

4.2 Optimizing the joint detection mathematical model

On the premise of ensuring the detection effect, in order to make the preparation and use of the kit easier, the above 16 phage clones were streamlined and screened. Methods as below:

All independent serum samples were randomly sampled and numbered 2000 times using the random forest method (Random Forest) by dige R software, and the screening ability of these 16 phage clones was sorted from high to low; according to the sorting results, the top 16 and 15 were selected in turn. 1, 14, until the first 3 phage clones, input their reactivity values into the Bayesian binary regression, and found that the combined number of phage clones was the least, that is, only the first five phage clones (phage 1009 clone, phage clone No. 174, phage clone No. 149, phage clone No. 396, and phage clone No. 95), the sensitivity and specificity of the joint screening were similar to those of the joint screening of 16 phage clones, and the sensitivity and specificity were respectively 90.0% and 96.7%, as shown in Figure 4, the area under the ROC curve of the joint monitoring of 16 phage clones was 0.937, and the area under the ROC curve of the joint detection of 5 phage clones was 0.940. After sequencing, the sequences of the cDNA inserts of the first five phage clones are shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, and SEQ ID No.9, corresponding to the The amino acid sequences of the encoded colorectal cancer-specific antigen peptides are shown in SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, and SEQ ID No.10.

Therefore, the final joint detection model is a Bayesian binary regression model established by summarizing the response data of 5 different phage clones and 30 pairs of training groups (30 cancer patient sera and 30 healthy control sera). ), as shown in Figure 5.

Example 5 Application of verification group serum to test the combined screening effect of 5 phages

These five phage clones were respectively reacted with sera of 60 cases of colorectal cancer patients and 60 cases of control sera confirmed by the gold standard (pathological results), and the method was re-screened with the independent sera (Example 3.1). Input the detection value into the mathematical model established based on 30 pairs of independent blood samples in the training group to generate the probability that the tester suffers from colorectal cancer. The cut-off value is 0.5. If it is greater than or equal to 0.5, it is positive, and if it is less than 0.5, it is negative. The detection result is, The results are shown in Table 1. The sensitivity of ELISA detection method to detect colorectal cancer in the verification group was 90.0%, and the specificity was 91.7%.

Table 1 ELISA detection results

Example 6 Comparison of combined detection method of phage clone and detection method of tumor marker CEA In order to compare the combined detection method of phage clone with the current detection method of tumor marker CEA. Summarize the data of 60 pairs of colorectal cancer serum and control serum used in serological verification, and draw the ROC curve to verify the significance of the screening according to the CEA screening method, single phage clone screening method, and phage clone combined detection method, and calculate the ROC curve The results are shown in Table 2. The closer the area under the ROC curve is to 1, the higher the sensitivity of the screening method, and the higher the specificity, the better the detection effect. The results in Table 2 show that the ability to distinguish colorectal cancer using only a single phage clone is superior to the traditional CEA detection method, and the combined detection method of 5 phage clones is more accurate. Therefore, the phage display peptide detection method can provide a more effective means of discrimination for patients with early colorectal cancer.

Table 2 Comparison of the area under the curve of the ROC drawn by different screening methods

Area under the ROC curve 95% confidence interval P value CEA 0.776 0.689-0.862 P<0.001 Phage No. 1009 0.895 0.839-0.951 P<0.001 Phage 149 0.824 0.749-0.898 P<0.001 Phage No. 174 0.893 0.835-0.952 P<0.001 Phage No. 396 0.856 0.790-0.922 P<0.001 Phage 95 0.833 0.762-0.905 P<0.001 Joint screening of 5 phages 0.973 0.949-0.997 P<0.001

Example 7 Operation of Colorectal Cancer-specific Autoantibody ELISA Kit

7.1 Preparation of colorectal cancer-specific antigen ELISA plate

Dilute T7TAILER monoclonal antibody at 1:1000 dilution with 1×PBS (PH7.4), coat 100 μl per well on a 96-well ELISA plate, and shake gently overnight at 4°C on a shaker. 1× washing solution, 300 μl per well, wash four times, about 1 minute each time, and pat dry. 2%BSA/PBS200μl at room temperature (26°C) to block for 2 hours.

1× washing solution, 300 μl per well, wash four times, about 1 minute each time, and pat dry. Add 100 μl of No. 95, No. 149, No. 174, No. 396 and No. 1009 diluted 1:5 with 1% BSA, and add 2 wells for each specimen. In each experiment, bacteriophage No. 50 without insert fragments was added as negative control and 2 wells of blank control. Incubate at room temperature for 2 hours. The liquid in the wells was discarded and patted dry. At this time, due to the antigen-antibody reaction, the tumor-specific antigen was successfully immobilized on the microtiter plate.

7.2 ELISA detection of colorectal cancer patients

Add 100 μl 1% BSA 1:500 diluted colorectal cancer patient plasma to each phage sample, and incubate at room temperature for 1 hour. Wash the plate with washing solution, 300 μl per well, wash four times, about 1 minute each time, and pat dry. Add 100 μl of 1:10000 diluted HRP-coupled goat anti-human IgG to each well and incubate at room temperature for 1 hour. Wash the plate with washing solution, 300 μl per well, wash four times, about 1 minute each time, and pat dry. Add 2 drops of chromogenic reagents A and B each at room temperature to each well, mix well, incubate at 37°C for 5 minutes, and add 25 μl of stop solution. Immediately detect with a microplate reader, take a wavelength of 450nm, first use a blank well to zero, then read the OD value of each well, and record the results.

7.3 Result Judgment

Each ELISA is calculated and judged according to the following formula: after zeroing the blank well, the average OD value of the sample/the average OD value of the negative control, the reaction results of the serum and 5 phage clones are substituted into the joint screening model, and the cut-off is set. The value is 0.5, when the result is ≥0.5, it is judged as positive; if the result is <0.5, the result is judged as negative. The kit judges that the serum to be tested may be the serum of patients with early colorectal cancer.

The five colorectal cancer-specific antigen peptide expression vectors of the present invention effectively express colorectal cancer-specific antigen peptides, are coated on a microtiter plate, and have the ability to detect autoantibodies produced by tumor stimulation in the serum of colorectal cancer patients. So as to achieve the purpose of early detection and screening of colorectal cancer patients. Compared with other current methods, it has the characteristics of minimally invasive, simple, economical, high sensitivity and specificity, and is convenient for screening of community population, so as to achieve the purpose of early detection of colorectal cancer.

Claims (2)

1. A colorectal cancer-specific antigenic peptide, characterized in that the amino acid sequence of the colorectal cancer-specific antigenic peptide is as shown in SEQ ID No.10. 2. An expression vector for expressing the colorectal cancer-specific antigen peptide according to claim 1, characterized in that, it is composed of T7 phage and a cDNA fragment encoding the colorectal cancer-specific antigen peptide, and the sequence of the cDNA fragment is as follows: Shown in SEQ ID No.9.

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