人朊蛋白基因的克隆表达及抗血清的制备

发布时间：2018-05-06 08:09

本文选题：人 + 朊蛋白基因　；参考：《甘肃农业大学》2006年硕士论文

【摘要】： 以人外周血液提取的总DNA为模板,PCR扩增人朊蛋白编码基因(PRNP)的ORF,克隆至pMD18T载体,构建重组质粒HoPRNP-T,用DNAstar软件与GenBank上发表的序列M13899进行同源性分析,结果发现核苷酸序列同源性为99.6㳠,氨基酸序列同源性99.8%。以HoPRNP-T质粒为模板,扩增出编码人成熟朊蛋白(mature prion protein,mPrP)的基因片断,与pET30a(+)表达载体连接,构建出表达人mPrP的重组质粒pET-homPrP。经酶切鉴定后电转化pET-homPrP至宿主菌E. coli BL21 (DE3)中,IPTG诱导表达,SDS-PAGE显示表达产物为分子量为约30ku的融合蛋白。超声波裂解重组菌体后用Ni-NTA亲和层析法纯化融合蛋白,以SDS-PAGE检测纯化的表达产物,表明亲和层析法纯化的融合蛋白纯度可达到90%。再以SAF70进口单抗为检测抗体,Western-blotting显示融合蛋白具有良好的反应原性。以纯化的融合mPrP作为免疫原,以含有pET30a(+)空载体的E. coli BL21 (DE3)的菌体蛋白作为对照免疫原,分别与弗氏完全佐剂1:1混合乳化,按100μg/只剂量皮下分3点注射8周龄清洁级BALB/c雌性小鼠,在第3周和第5周以同法、同剂量弗氏不完全佐剂乳化的两种免疫原分别进行加强免疫,第3次免疫3天后摘眼球取血,用间接ELISA法检测抗血清的效价。结果表明抗人mPrP的血清效价为1:512; Western-blotting比较分析也显示此抗血清具有很强的特异性。此实验结果为进一步研究人朊蛋白结构和制备出相应的单克隆抗体奠定了基础。
[Abstract]:Using total DNA extracted from human peripheral blood as template, the ORF of human prion protein encoding gene was amplified by PCR and cloned into pMD18T vector. The recombinant plasmid HoPRNP-T was constructed. The sequence M13899 published on GenBank was analyzed by DNAstar software. The results showed that the homology of nucleotide sequence and amino acid sequence was 99.6 and 99.8 respectively. Using HoPRNP-T plasmid as template, the gene fragment encoding human mature prion protein mPrPwas amplified and ligated with pET30a () expression vector. The recombinant plasmid pET-homPrPexpressing human mPrP was constructed. SDS-PAGE showed that the expressed product was a fusion protein with a molecular weight of about 30ku in the electroporation of pET-homPrP to the host strain E. coli BL21 (DE3) by restriction endonuclease digestion. The fusion protein was purified by Ni-NTA affinity chromatography after ultrasonic lysis, and the expressed product was detected by SDS-PAGE. The purity of the fusion protein purified by affinity chromatography could reach 90%. Western blotting showed that the fusion protein had good reactivity. The purified fusion mPrP was used as immunogen, and the bacterial protein containing pET30a () empty vector, E. coli BL21 (DE3), was used as control immunogen, and emulsified with Freund's complete adjuvant 1:1, respectively. Clean grade BALB/c female mice of 8 weeks old were injected subcutaneously with 100 渭 g / g of 100 渭 g / L subcutaneously. The two immunogens emulsified by Freund's incomplete adjuvant were immunized with the same method at the 3rd week and the 5th week, respectively. The eyeball blood was taken out 3 days after the third immunization. The titer of antiserum was detected by indirect ELISA method. The results showed that the titer of antihuman mPrP was 1: 512, and the Western-blotting analysis showed that the antiserum had a strong specificity. The results laid a foundation for the further study of the structure of human prion protein and the preparation of corresponding monoclonal antibodies.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346

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