生物技术通讯
生物技術通訊
생물기술통신
LETTERS IN BIOTECHNOLOGY
2014年
6期
800-803
,共4页
人乳头瘤病毒18型L1蛋白%包涵体%可溶性蛋白%纯化
人乳頭瘤病毒18型L1蛋白%包涵體%可溶性蛋白%純化
인유두류병독18형L1단백%포함체%가용성단백%순화
human papillomavirus 18 L1 protein%inclusion body%soluble protein%purification
目的：原核表达系统表达人乳头瘤病毒18型（HPV18）L1蛋白，建立包涵体和可溶性表达的L1蛋白的纯化方法。方法：构建重组表达质粒pGEX-4T-1-HPV18 L1，在大肠杆菌BL21中以包涵体和可溶性方式表达HPV18 L1蛋白。通过超声波破碎菌体、洗涤包涵体、碱变性、透析复性和谷胱甘肽（GST）琼脂糖凝胶4B亲和层析纯化包涵体蛋白；在菌体中加入三磷酸腺苷（ATP）和3.5 mol/L尿素孵育后，GST 4B亲和层析纯化可溶性蛋白，凝血酶酶切。SDS-PAGE和Western印迹鉴定表达和纯化产物。结果：SDS-PAGE结果表明，HPV18 L1蛋白以包涵体和可溶性方式在大肠杆菌BL21内高效表达，均产生相对分子质量约为86000的HPV18 L1-GST融合蛋白。Western印迹结果显示，包涵体纯化后获得的融合蛋白降解条带较多；而可溶性蛋白纯化后获得的融合蛋白未降解，凝血酶酶切后得到HPV18 L1蛋白，可与HPV18 L1蛋白单克隆抗体结合。结论：采用原核系统表达了HPV18 L1-GST融合蛋白，分别建立了包涵体和可溶性蛋白的纯化方法，获得HPV18 L1蛋白，为其进一步应用奠定了基础。
目的：原覈錶達繫統錶達人乳頭瘤病毒18型（HPV18）L1蛋白，建立包涵體和可溶性錶達的L1蛋白的純化方法。方法：構建重組錶達質粒pGEX-4T-1-HPV18 L1，在大腸桿菌BL21中以包涵體和可溶性方式錶達HPV18 L1蛋白。通過超聲波破碎菌體、洗滌包涵體、堿變性、透析複性和穀胱甘肽（GST）瓊脂糖凝膠4B親和層析純化包涵體蛋白；在菌體中加入三燐痠腺苷（ATP）和3.5 mol/L尿素孵育後，GST 4B親和層析純化可溶性蛋白，凝血酶酶切。SDS-PAGE和Western印跡鑒定錶達和純化產物。結果：SDS-PAGE結果錶明，HPV18 L1蛋白以包涵體和可溶性方式在大腸桿菌BL21內高效錶達，均產生相對分子質量約為86000的HPV18 L1-GST融閤蛋白。Western印跡結果顯示，包涵體純化後穫得的融閤蛋白降解條帶較多；而可溶性蛋白純化後穫得的融閤蛋白未降解，凝血酶酶切後得到HPV18 L1蛋白，可與HPV18 L1蛋白單剋隆抗體結閤。結論：採用原覈繫統錶達瞭HPV18 L1-GST融閤蛋白，分彆建立瞭包涵體和可溶性蛋白的純化方法，穫得HPV18 L1蛋白，為其進一步應用奠定瞭基礎。
목적：원핵표체계통표체인유두류병독18형（HPV18）L1단백，건립포함체화가용성표체적L1단백적순화방법。방법：구건중조표체질립pGEX-4T-1-HPV18 L1，재대장간균BL21중이포함체화가용성방식표체HPV18 L1단백。통과초성파파쇄균체、세조포함체、감변성、투석복성화곡광감태（GST）경지당응효4B친화층석순화포함체단백；재균체중가입삼린산선감（ATP）화3.5 mol/L뇨소부육후，GST 4B친화층석순화가용성단백，응혈매매절。SDS-PAGE화Western인적감정표체화순화산물。결과：SDS-PAGE결과표명，HPV18 L1단백이포함체화가용성방식재대장간균BL21내고효표체，균산생상대분자질량약위86000적HPV18 L1-GST융합단백。Western인적결과현시，포함체순화후획득적융합단백강해조대교다；이가용성단백순화후획득적융합단백미강해，응혈매매절후득도HPV18 L1단백，가여HPV18 L1단백단극륭항체결합。결론：채용원핵계통표체료HPV18 L1-GST융합단백，분별건립료포함체화가용성단백적순화방법，획득HPV18 L1단백，위기진일보응용전정료기출。
Objective: To express human papillomavirus type 18(HPV18) L1 protein in a prokaryotic system, and establish the purification methods of HPV18 L1 inclusion body and soluble protein. Methods: The prokaryotic expression vector pGEX-4T-1-HPV18 L1 was constructed and transformed into E.coli BL21 cell and induced by IPTG to express HPV18 L1 protein in inclusion body and soluble forms. Inclusion body purification was conduct?ed in the following order: sonic disruption, washing, alkaline denaturation, dialysis renaturation and glutathione Sep?harose 4B(GST 4B) affinity chromatography. When the soluble protein purified, adding ATP and 3.5 mol/L urea to the lysate, and then the GST 4B affinity chromatography and thrombin cleavage was conducted. The expressed and purified products were identified by SDS-PAGE and Western blot. Results: SDS-PAGE results showed that HPV18 L1 protein was expressed efficiently in E.coli BL21 in inclusion body and soluble forms, the fusion pro?tein of Mr 86 000 was successfully induced. Western blot results showed that HPV18 L1-GST fusion protein of in?clusion body purification degenerated; the fusion protein of soluble protein purification was intact, and then HPV18 L1 protein was obtained by thrombin cleavage which has specific reaction with HPV18 L1 monoclonal anti?body. Conclusion: The fusion protein of HPV18L1-GST was prokaryotic expressed successfully; the study estab?lished the purification methods of inclusion body and soluble proteins and obtained HPV18 L1 protein which lay the foundation for application of HPV18 L1 protein further.
