本文選題：人類免疫缺陷病毒1型 + gp120��； 參考：《西北大學(xué)》2006年碩士論文

【摘要】：人類免疫缺陷病毒(HIV)是引起艾滋病的病原體,分為兩型:HIV-1和HIV-2,主要為HIV-1。它和一般的病毒有明顯的差別,首先是其基因組相對(duì)復(fù)雜,編碼更多的蛋白質(zhì)來(lái)調(diào)控病毒潛伏、激活、轉(zhuǎn)錄與復(fù)制等過(guò)程,其次是它在宿主個(gè)體內(nèi)變異迅速。鑒于HIV-1包膜糖蛋白gp120和gp41是HIV-1的主要抗原成分,是檢測(cè)HIV抗體診斷試劑盒的重要組成成分,也是制備抗gp120和gp41單克隆抗體必需的免疫原,應(yīng)用廣泛,意義十分重大。本研究利用分子生物學(xué)手段,將gp120和gp41的編碼基因分別重組到畢赤酵母及大腸桿菌的表達(dá)載體中,并成功地進(jìn)行了蛋白表達(dá)。 外膜糖蛋白gp120的編碼基因的克隆和在畢赤酵母中的表達(dá)。以質(zhì)粒pHXB2-gp160為模板進(jìn)行PCR,擴(kuò)增出gp120抗原的全長(zhǎng)DNA(gp120L)和短片段DNA(gp120S),構(gòu)建了酵母表達(dá)載體pPICZ α A-gp120L和pPICZ α A-gp120S。將重組質(zhì)粒線性化后轉(zhuǎn)化到畢赤酵母GS115細(xì)胞株,使gp120基因同源重組整合到畢赤酵母的染色體上,經(jīng)過(guò)選擇培養(yǎng)基、PCR鑒定、Zeocin抗性篩選后,將得到的陽(yáng)性菌株進(jìn)行甲醇誘導(dǎo)表達(dá),利用SDS-PAGE和Western blot分析表達(dá)產(chǎn)物,結(jié)果表明gp120L基因沒(méi)有得到表達(dá),gp120S基因在酵母胞內(nèi)有微量表達(dá),表達(dá)產(chǎn)物為19 kD。 跨膜糖蛋白gp41短片段基因在大腸桿菌中的表達(dá)、純化及初步應(yīng)用。同樣以pHXB2-gp160為模板進(jìn)行PCR,擴(kuò)增出gp41抗原短片段DNA,構(gòu)建重組表達(dá)質(zhì)粒pET32a-gp41S,轉(zhuǎn)化大腸桿菌BL21(DE3)plys,用IPTG誘導(dǎo)表達(dá),表達(dá)產(chǎn)物經(jīng)過(guò)SDS-PAGE和Western blot分析證實(shí)35.5 kD的目的蛋白gp41S主要存在于包涵體中,凝膠掃描成像分析證實(shí)其表達(dá)量占菌體總蛋白的10%以上。包涵體經(jīng)過(guò)洗滌、溶解和復(fù)性后,純度可達(dá)90%以上,表明已獲得高純度的目的蛋白。用純化的重組gp41蛋白作為包被抗原,應(yīng)用間接ELISA法對(duì)4份HIV陽(yáng)性血清中的gp41抗體進(jìn)行檢測(cè),結(jié)果表明該純化蛋白具有一定的抗原活性,能特異性地與HIV抗體反應(yīng)。用該蛋白免疫小鼠,經(jīng)過(guò)三次免疫后抗體效價(jià)可達(dá)
[Abstract]:Human immunodeficiency virus (HIV) is the cause of AIDS, divided into two types: HIV-1 and HIV-2, mainly HIV-1. It is different from other viruses. Firstly, its genome is relatively complex, and it encodes more proteins to regulate the processes of virus latency, activation, transcription and replication, and then it mutates rapidly in host individuals. Since HIV-1 envelope glycoprotein gp120 and gp41 are the main antigen components of HIV-1, they are important components of HIV antibody diagnostic kit, and they are also the necessary immunogen to prepare monoclonal antibodies against gp120 and gp41. They are widely used and of great significance. In this study, the coding genes of gp120 and gp41 were recombined into Pichia pastoris and Escherichia coli by molecular biology, and the protein was successfully expressed. Cloning and expression of Encoding Gene of Outer membrane glycoprotein gp120 in Pichia pastoris. Plasmid pHXB2-gp160 was used as template to amplify the full-length DNA-gp120L of gp120 antigen and its short fragment, pPICZ 偽 A-gp120L and pPICZ 偽 A-gp120S. The yeast expression vectors pPICZ 偽 A-gp120L and pPICZ 偽 A-gp120Swere constructed. The recombinant plasmid was linearized and transformed into Pichia pastoris GS115 cell line. The homologous recombinant of gp120 gene was integrated into the chromosome of Pichia pastoris. The expression products were analyzed by SDS-PAGE and Western blot. The results showed that gp120L gene was not expressed in yeast cells and the expression product was 19kD. Expression, purification and preliminary application of transmembrane glycoprotein gp41 short fragment gene in Escherichia coli. Using pHXB2-gp160 as template, the short fragment of gp41 antigen was amplified, and the recombinant expression plasmid pET32a-gp41Swas constructed. The recombinant plasmid pET32a-gp41Swas transformed into E. coli BL21DDE3plys. the expression product was induced by IPTG. The result of SDS-PAGE and Western blot analysis confirmed that the target protein gp41S of 35.5 KD mainly existed in inclusion body. Gel scanning imaging analysis confirmed that its expression was more than 10% of the total bacterial protein. After washing, dissolving and renaturation, the purity of inclusion body was over 90%, which indicated that the target protein with high purity had been obtained. The purified recombinant gp41 protein was used as the coating antigen to detect the gp41 antibody in 4 HIV-positive sera by indirect Elisa. The results showed that the purified protein had a certain antigenic activity and could react specifically with HIV antibody. The antibody titers of mice immunized with this protein were up to three times.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：Q789;R392

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