本文選題：人 + 朊蛋白基因　； 參考：《甘肅農(nóng)業(yè)大學(xué)》2006年碩士論文

【摘要】： 以人外周血液提取的總DNA為模板,PCR擴(kuò)增人朊蛋白編碼基因(PRNP)的ORF,克隆至pMD18T載體,構(gòu)建重組質(zhì)粒HoPRNP-T,用DNAstar軟件與GenBank上發(fā)表的序列M13899進(jìn)行同源性分析,結(jié)果發(fā)現(xiàn)核苷酸序列同源性為99.6㳠,氨基酸序列同源性99.8%。以HoPRNP-T質(zhì)粒為模板,擴(kuò)增出編碼人成熟朊蛋白(mature prion protein,mPrP)的基因片斷,與pET30a(+)表達(dá)載體連接,構(gòu)建出表達(dá)人mPrP的重組質(zhì)粒pET-homPrP。經(jīng)酶切鑒定后電轉(zhuǎn)化pET-homPrP至宿主菌E. coli BL21 (DE3)中,IPTG誘導(dǎo)表達(dá),SDS-PAGE顯示表達(dá)產(chǎn)物為分子量為約30ku的融合蛋白。超聲波裂解重組菌體后用Ni-NTA親和層析法純化融合蛋白,以SDS-PAGE檢測純化的表達(dá)產(chǎn)物,表明親和層析法純化的融合蛋白純度可達(dá)到90%。再以SAF70進(jìn)口單抗為檢測抗體,Western-blotting顯示融合蛋白具有良好的反應(yīng)原性。以純化的融合mPrP作為免疫原,以含有pET30a(+)空載體的E. coli BL21 (DE3)的菌體蛋白作為對照免疫原,分別與弗氏完全佐劑1:1混合乳化,按100μg/只劑量皮下分3點(diǎn)注射8周齡清潔級(jí)BALB/c雌性小鼠,在第3周和第5周以同法、同劑量弗氏不完全佐劑乳化的兩種免疫原分別進(jìn)行加強(qiáng)免疫,第3次免疫3天后摘眼球取血,用間接ELISA法檢測抗血清的效價(jià)。結(jié)果表明抗人mPrP的血清效價(jià)為1:512; Western-blotting比較分析也顯示此抗血清具有很強(qiáng)的特異性。此實(shí)驗(yàn)結(jié)果為進(jìn)一步研究人朊蛋白結(jié)構(gòu)和制備出相應(yīng)的單克隆抗體奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R346

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