The Investigation Of CypA By Liquid Chromatography-mass Spectrometry Technology And Evaluation Of Its Monoclonal Antibody In The Diagnosis Of Colorectal Cancer

Colorectal cancer,also known as colorectal cancer(CRC),Colorectal cancer(CRC)is the third most common cancer worldwide.The estimated numbers of new CRC cases and CRC deaths in the United States in 2014 were approximately 135,000 and 50,000,respectively.The life-time risk of being diagnosed with CRC is approximately 5% in the Western world.Stage at diagnosis is the most important prognostic factor for CRC,with a 5-year survival of approximately 90% for earlystage disease without regional or distant metastases but only around 10% when distant metastases are present.3 Unlike in many other Western countries,CRC incidence rates have declined in China since the mid-1980 s,particularly for late-stage disease.Although the reasons for this decline are not clear,a concomitant increase in CRC screening may be an important factor.Individuals with a strong family history of CRC,inflammatory bowel disease,and rare genetic conditions such as familial adenomatous polyposis and Lynch syndrome are at increased risk of CRC and should be referred for specialized care and surveillance.However,most CRC cases develop in the so-called average-risk population without any of these known risk factors.In the general population,age is the most important risk factor for CRC,with 90% of cases developing in individuals more than 50 years of age.Epidemiologic studies indicate that elements of Western lifestyle may influence the risk of CRC,but the effect of lifestyle modifications is still unclear.Factors that have been linked to a risk increase include high intake of red and processed meat,smoking,excessive alcohol consumption,and obesity,whereas physical activity and the use of aspirin have been associated with decreased risk of CRC.In the absence of well-defined options for primary CRC prevention,screening is currently the most widely accepted approach to reduce CRC burden.In recent years,many researchers have used molecular biology techniques such as proteomics to study tumor markers of colorectal cancer,and currently known related molecular markers such as KRAS(predictive marker for anti-EGFR antibody treatment),B-Raf V600E(a biomarker of anti-EGFR antibody resistance),PIK3 CA,ERCC-1(potentially predictive marker of oxaliplatin),PTEN(short-term progressionfree survival in patients with CRC),COX-2,Ezrin.The extensive use of proteomics has led to a deepening understanding of cancer.There are four parts in this research: Part Ⅰ: Establishment of differential protein profile of TNM stage Ⅲ colorectal cancer and adjacent tissuesMethod:1.The specimens were 20 pairs of colorectal adenocarcinoma and matched adjacent tissues,(adjacent tissues from the cancer tissue> 10 cm.)Specimens were not receiving any treatment,and confirmed by pathology for TNM stage Ⅲ adenocarcinoma.2.Preparation of a proteolytic polypeptide mixture.3.Through the LTQXL ion hydrazine mass spectrometry on the two-dimensional chromatography elution of the peptide detection.4.Compare the database,analyze the biological information in the mass spectrometry data,and establish the differentially expressed protein spectrum of colorectal adenocarcinoma.Result:1.Analyze and compare the protein spots obtained from the experiment: 567 tumor tissues;434 adjacent tissues;we obtained 30 differentially expressed proteins in colorectal adenocarcinoma tissues.2.Up-regulated expressed proteins(20): Cyp A,ATP5 B,TPM4,FGA,GSTP1,HIST1H3 J,IGHA1,SERPINH1,TUBB3,IGHA1,TPM3,CKAP4,IGHA2,POSTN,FGB,FGG,PKM2,HIST1H2 AE,MYH10,and TUBB.3.Down-regulated expression proteins(10): SYNM,MYH11,PKM2,KRT10,FLNC,H2 AFX,CAP1,MYL6,TUBB2 A,TLN1.Part Ⅱ: Cyp A gene amplification,vector construction and induction of expression of fusion proteinMethod:1.According to Gen Bank-nucleotide,chemically synthesized Cyp A gene sequence,polymerase chain reaction amplification of the target gene Cyp A.2.The amplified Cyp A was ligated with the vector and the prokaryotic expression vector p ET28a/Cyp A was constructed and transformed into E.coli BL21 to obtain the expression strain BL21(DE3)(p ET28a/Cyp A),which induced the expression of Cyp A.3.The target protein was purified by affinity chromatography on nickel agarose,and the target protein was detected by SDS-PAGE.4.Western blot analysis to identify the protein of target.Result:1.The prokaryotic expression vector p ET28a/Cyp A was successfully constructed and confirmed by DNA sequencing and database comparison.2.After the transformation of BL21,induced expression of transformed strains,protein purification,SDS-PAGE and Western blotting identification,purified Cyp A protein was obtained.Part Ⅲ: Preparation,purification and identification of Cyp A monoclonal antibodyMethod:1.Purified Cyp A fusion protein was injected subcutaneously to immunize mice.The spleen cell-myeloma cell fusion of the immunized mouse was prepared.2.Selective medium HAT cultured fusion cells,ELISA screening of fusion cell supernatant,monoclonal purification of positive hybridoma cell lines;3.The positive hybridoma cell strain was injected into the abdominal cavity of the mice to prepare ascites,and the antibody titer of ascites was detected by ELISA.4.ELISA detection of monoclonal antibody subtypesResult:1.Obtained an anti-human-Cyp A hybridoma cell strain: stable secretion of antihuman-Cyp A monoclonal antibody;2.Ascites antibody titer of 1:160000,antibody purity 95%.Part Ⅳ: Expression of Cyp A in colorectal cancer and its relationship with clinicopathological parametersMethod:1.Thirty pairs of colorectal adenocarcinoma tissue specimens were classified according to pathological TNM staging,and a database of clinical pathological data was established;2.Immunohistochemical assay to evaluate the expression of the target protein Cyp A in paired tumor tissues and paracancerous tissues;3.Analyze the correlation between the difference in the expression of the target protein Cyp A and the clinicopathological parameters of the tumor patients The expression of Cyp A in colorectal cancer tissues and adjacent tissues is detected by immunohistochemical method.Result:1.30 pairs of colorectal adenocarcinoma patients with tumor tissue and adjacent tissues have the expression of the target protein Cyp A,and by immunohistochemistry results,the expression of the target protein in the tumor tissue is higher than the adjacent tissue.2.In colorectal adenocarcinoma,differentiation and lymph node metastasis are related to the expression of the target protein Cyp A.Conclusions1.In this study,two-dimensional liquid chromatography-mass spectrometry analysis and database comparisons were performed to obtain differentially expressed protein profiles in tumor tissues and adjacent tissues of colorectal adenocarcinoma patients.2.The prokaryotic expression vector p ET28a/Cyp A was successfully constructed,and the hybridoma cell line stably expressing the target protein Cyp A was obtained.The monoclonal antibody of the target protein Cyp A was prepared and purified,which laid the foundation for the subsequent analysis and application of the Cyp A protein and clinical data.3.Immunoblotting and immunohistochemistry confirmed that the expression of Cyp A in colorectal cancer tissues was significantly higher than that in paracancerous tissues.The results of two-dimensional liquid chromatography-mass spectrometry analysis were confirmed,and previously established colorectal cancer tumor tissues and cancer were identified.The reliability of differential protein profiles in para-organisms.4.Analysis of the relationship between the expression level of Cyp A protein and lymph node metastasis and TNM stage provides a reference for the diagnosis and treatment of colorectal cancer..