HIV-1外膜蛋白gp120在酵母Pichia pastoris中的表达  

Expression of HIV-1 gp 120 in methylotrophic Pichia Pastoris

作　　者：冯劼 朱娟莉 董兆麟[1] 陈超[1,3] 

机构地区：[1]西北大学生命科学学院,陕西西安710069 [2]陕西北美基因股份公司,陕西西安710069 [3]国家微检测系统工程技术研究中心

出　　处：《西北大学学报（自然科学版）》2009年第5期801-804,813,共5页Journal of Northwest University（Natural Science Edition）

基　　金：国家高技术研究发展计划基金资助项目(2005AA205220)

摘　　要：目的用巴斯德毕赤酵母系统表达HIV外膜糖蛋白gp 120。方法将从HIV-1型国际标准株pHXB2-gp 160的基因序列中扩增出的gp 120分子的长片段基因(1 419 bp)和短片段基因(417bp)分别克隆入真核表达载体pPICZαA与pPICZB中,以电穿孔法转化酵母GS 115。用YPDS-zeo平板筛选重组子、PCR方法检测整合到酵母菌GS 115基因组中的gp 120片段基因,经甲醇诱导表达后,SDS-PAGE和免疫印迹分析表达产物。结果诱导后gp 120短片段多肽在GS 115中少量表达,在诱导表达24 h重组蛋白表达量及抗原性达到最高,表达产物被降解,具有良好的抗原特异性。诱导后gp 120长片段多肽未被GS 115所表达。结论在巴斯德毕赤酵母中成功表达gp 120分子长片段需要优化gp 120基因,为制备HIV-1的诊断抗原和基因工程重组疫苗打下基础。Aim To express HIV-1 protein gp 120 in Pichia Pastoris. Methods A long fragment （ 1 419 bp） and a short fragment （417 bp） of gp 120 gene were amplified from HIV-1 international standard strain pHXB2-gp 160, and were sub-cloned into eukaryotic expression vector pPICZaA and pPICZB. The constructed plasmid was transformed into yeast GSll5 by electroporation. The recombinant transformants were selected by YPDS plates, and PCR was used to test the insert. The expression in yeast was induced by the addition of methanol and was analyzed by SDS-PAGE and Western blot. Results The gp 120 short peptide was expressed in GS 115. At 24 hours induction time, its expression level and antigenecity are the highest. The expressed product was truncated and showed excellent antigenic specialty. The gpl20 long peptide was not expressed by GS115. Conclusion Gene optimization should be performed to express successfully the gp 120 long DNA fragment in Pichia Pastorls. the research is useful for the production of diagnostic reagents and genetically engineered vaccine of HIV-1.

关 键 词：人免疫缺陷病毒I型 GP 120 基因克隆 蛋白表达 毕赤酵母 

分 类 号：Q513.2[生物学—生物化学]