LP-PLA2的原核表達(dá)、抗體制備及表位分析
[Abstract]:Lipoprotein associated phospholipase A 2 (lipoprotein associated phospholipase A 2) belongs to VIIA type phospholipase A 2, which can predict the risk of atherosclerosis and stroke associated with coronary events. At present, the LP-PLA2 clinical diagnostic reagent mainly depends on imported from abroad, and the cost is expensive, which limits the application and popularization of the test project in China. Therefore, it is particularly important to develop LP-PLA2 in vitro immunodiagnostic reagent. Objective to obtain human lipoprotein associated phospholipase A2 protein and prepare the corresponding antibody, and to verify the antigenicity of the epitope peptide predicted by bioinformatics, so as to lay a foundation for the development of LP-PLA2 immunological diagnostic reagent in vitro. Methods the total RNA, was extracted from differentiated THP-1 cells by Trizol method and the target fragment LP-PLA2; was amplified by RT-PCR. The prokaryotic expression plasmid was constructed by molecular cloning technique. E. coli BL21 (DE3) was transformed into Escherichia coli (DE3), and the expression conditions were optimized. The target protein was purified and the label was removed. The target protein was immunized against New Zealand white rabbits, and the polyclonal antibody against LP-PLA2 was prepared. LP-PLA2 epitopes were predicted by various bioinformatics methods and their antigenicity was identified with purified LP-PLA2 polyclonal antibodies. Result 1. PET-28a ()-LP-PLA2,PGEX-4T-2-LP-PLA2,pCold TF-LP-PLA2 and other recombinant expression plasmids were successfully constructed. 2. After IPTG induced expression, SDS-PAGE electrophoresis showed that LP-PLA2 was not expressed in PET-28a ()-LP-PLA2 expression vector, but was expressed in a small amount in PGEX-4T-2-LP-PLA2 expression vector, and mainly in the form of inclusion body. In the pCold TF-LP-PLA2 expression vector, the fusion protein with high expression level was obtained, and the main supernatant was found. 3. The recombinant protein of pCold TF-LP-PLA2 was purified by nickel column affinity chromatography. The recombinant protein was digested with HRV-3C prolease and purified with blue gel. The purity of the recombinant protein was about 90%. The protein with a molecular weight of about 45kD was in line with the expectation, and the protein could be recognized by the LP-PLA2 polyclonal antibody on the market. It was proved that the purified LP-PLA2 target protein was obtained. 4. The polyclonal antibody against LP-PLA2 was prepared. The titer of the antibody was 1: 5.12 脳 10 ~ 6, Western blot. The specificity of the antibody was high. 5. The three-segment epitope peptides of LP-PLA2 were predicted by bioinformatics software. The results of antigen-antibody immunoreactivity in vitro confirmed that all three epitope peptides had good antigenicity. Conclusion High purity LP-PLA2 protein was successfully expressed and purified, and LP-PLA2 polyclonal antibody with high titer and purity was prepared. The three-segment epitope peptide of LP-PLA2 was predicted by bioinformatics, and its antigenicity was confirmed. It can be used to prepare polyclonal and monoclonal antibodies. This study laid a foundation for the development of LP-PLA2 in vitro diagnostic reagent.
【學(xué)位授予單位】:重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】:碩士
【學(xué)位授予年份】:2011
【分類號(hào)】:R392.1
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