【摘要】：目的:利用基因工程技術(shù),分別構(gòu)建含HBV/RNAse H1及H2基因的原核表達(dá)載體pGSTag,并轉(zhuǎn)化到大腸桿菌,誘導(dǎo)表達(dá)出目的蛋白后,用原核表達(dá)產(chǎn)物作為抗原,制備單克隆抗體,并對(duì)抗單克隆抗體血清型進(jìn)行初步分析鑒定,為臨床應(yīng)用奠定基礎(chǔ)。 方法:本研究采用搖瓶發(fā)酵,將重組質(zhì)粒pGSTag/HBV/RNAseH1及H2分別轉(zhuǎn)化BL21(DE3)codon plus(?)-RP、HB101、BL21(DE3)PlysS、G1727、DH5α、BL21大腸桿菌。同時(shí)用不同的培養(yǎng)基(LB、SOB、SOC、TB、2YT、M9)、不同的初始菌濃度(即OD為0.2,0.4,0.6,0.8,1.0,1.2)、不同的誘導(dǎo)時(shí)間(2.0h,4.0h,6.0h)、初始pH(6.0,6.5,7.0,7.5,8.0,8.5)以及不同的IPTG誘導(dǎo)濃度(0.1,0.3,0.6,1.0,2.0,3.0mmol/L),分別表達(dá)重組蛋白,并將重組菌與空白菌作生長(zhǎng)曲線對(duì)比,收集菌體,初步純化,菌液(0.5ml)經(jīng)離心洗滌后,進(jìn)行SDS-PAGE分析,并用FR-200紫外可見(jiàn)分析裝置、復(fù)日smart view 2001生物電泳圖象分析系統(tǒng)分析pGSTag/HBV-RNAseH1及H2蛋白表達(dá)量,同時(shí)進(jìn)行Westerblot印跡分析。并以
[Abstract]:The invention aims to construct a prokaryotic expression vector pGSTag containing the HBV/ RNAse H1 and the H2 gene by using the genetic engineering technology, and is transformed into E. coli to induce the expression of the target protein, and the prokaryotic expression product is used as an antigen to prepare the monoclonal antibody. And a preliminary analysis and identification of the monoclonal antibody serotype is carried out to lay a foundation for clinical application. Methods: The recombinant plasmid pGSTag/ HBV/ RNAseH1 and H2 were transformed into BL21 (DE3) coon plus (?)-RP, HB101, BL21 (DE3) PlysS, G1727 and DH5 by shake flask fermentation. BL21. coli. At the same time, different culture media (LB, SOB, SOC, TB, 2YT, M9), different initial bacterial concentrations (i.e., OD of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2), different induction times (2.0 h, 4.0 h, 6.0 h), initial pH (6.0, 6.5, 7.0, 7.5, 8.0, 8.5), and different IPTG-induced concentrations (0.1, 0.3, 0.6, 1.0, 2.0, 3.0 mmol/ L), The recombinant protein was expressed separately, and the recombinant bacteria were compared with the blank fungus as the growth curve. The cells were collected, and the bacterial liquid (0.5 ml) was purified by centrifugation. The SDS-PAGE analysis was carried out. and the expression of the H2 protein is carried out at the same time, Bl
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類(lèi)號(hào)】：R392

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相關(guān)期刊論文 前3條

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本文編號(hào)：2442772