HIV-1 CN54 Pol P51抗原高效表達、純化、復性及在抗體檢測中的應用

侯爵，孫靜，徐智勇，范文玲，張怡軒，劉勇，郝彥玲

1 沈陽藥科大學 生命科學與生物制藥學院，沈陽 110016 2 中國疾病預防控制中心 性病艾滋病預防控制中心 傳染病防治國家重點實驗室，北京 100050

HIV-1 CN54 Pol P51抗原高效表達、純化、復性及在抗體檢測中的應用

侯爵1,2，孫靜2，徐智勇2，范文玲2，張怡軒1，劉勇2，郝彥玲2

1 沈陽藥科大學 生命科學與生物制藥學院，沈陽 110016 2 中國疾病預防控制中心 性病艾滋病預防控制中心 傳染病防治國家重點實驗室，北京 100050

為獲得可溶性高純度HIV-1中國株CN54 Pol P51抗原，將攜帶CN54 pol p51基因的重組質粒pTHioHisA51轉化大腸桿菌BL21 codonplus-RIL，用IPTG進行誘導表達。用Chelating Sepharose FF-Ni親和層析柱及DEAE Sepharose Fast Flow陰離子交換層析柱純化目的蛋白，采用透析復性法得到可溶性抗原，Western blotting檢測目的蛋白。用純化的P51抗原蛋白標記辣根過氧化物酶及包被酶標板進行雙抗原夾心法ELISA檢測。結果顯示P51以包涵體的形式表達，表達量占菌體總蛋白的50%，經兩步層析和透析復性，目的蛋白純度大于95%。Western blotting和雙抗原夾心法ELISA檢測均顯示了良好的靈敏度和特異性。本研究可以為HIV-1疫苗研究和開發檢測試劑提供支持。

HIV-1 CN54，逆轉錄酶P51，純化，復性，抗體檢測

Abstract:To obtain the pure and soluble P51 antigen of HIV-1 strain CN54, we transformed the Escherichia.coli strain BL21 codonplus-RIL with recombinant plasmid pTHioHisA51 which carries a gene encoding the Polymerase(Pol)P51 antigen of HIV-1 CN54 formerly, and induced protein expression by IPTG.We purified the recombinant protein with Chelating Sepharose FF-Ni and DEAE-Sepharose FF column chromatography, then renatured the recombinant protein by dialyzation.Purified protein was identified by Western blotting.We labeled and coated antigen P51 in a dual-antigen sandwich system, and tested it with serum samples from HIV-infected individuals.The results showed that P51 was expressed as inclusion body, and represented about 50% of total cellular protein.After purification and renaturation, the purity of P51 was up to 95%.Western blotting and sandwich ELISA demonstrated that recombinant P51 had good anti-HIV antibody specificity and sensitivity.The results suggested that recombinant HIV-1 P51 can be prepared as diagnostic reagent, and provides valuable support for HIV-1 detection and vaccine research.

Keywords:HIV-1 CN54, reverse transcriptase P51, purification, renaturation, antibody detection

HIV逆轉錄酶(RT)是pol基因編碼的一個異源二聚體蛋白，由P66和P51兩個亞單位組成，分子量約為117 kDa。逆轉錄酶在HIV生活史中具有逆轉錄酶、RNA酶H和整合酶活性，發揮著十分重要的作用。已有研究發現P66和P51作為抗原與HIV-1陽性血清具有很高的結合活性，P51抗原在進行 ELISA和Western blotting測定時，陽性檢出率與 HIV-1 Gp41相當，比HIV-1 Gag高[1-2]。P51是檢測HIV-1抗體檢測試劑中最常用的抗原，無論在初篩試劑還是確認試劑都要求有P51抗原的存在。……