中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2009年
5期
424-430
,共7页
王铮%李敬云%鲍作义%李韩平%庄道民%刘思杨%李林
王錚%李敬雲%鮑作義%李韓平%莊道民%劉思楊%李林
왕쟁%리경운%포작의%리한평%장도민%류사양%리림
HIV-1%gp120%糖基化蛋白
HIV-1%gp120%糖基化蛋白
HIV-1%gp120%당기화단백
HIV-1%gp120%Glycoprotein
目的 分析我国HIV-1主要亚型毒株的gp120区域的序列特征,并表达出gp120糖基化蛋白,为研究gp120的致病机制和设计疫苗奠定基础.方法 利用巢式PCR从来自于不同省份的5名HIV-1感染者外周血单个核细胞DNA中扩增全长gp120基因并测序,对序列特征进行分析,并将获得的gp120基因克隆入真核表达载体,体外表达获得gp120糖基化蛋白.结果 序列分析显示,此5条gp120序列分属HIV-1 Thai-B、BC重组和AE重组哑型,虽然亚型不同,但这些gp120序列在相同的位置都存在着一些保守的糖基化位点,且都具备相同的Furin蛋白酶第一切割位点,与参考序列比较发现,不同的HIV亚型序列中,除V3序列长度较为保守外,V1、V2、V4、V5各区域都有不同程度的缺失现象,与BC重组和AE重组亚型相比,Thai-B亚型V3的顶端环呈现出多种组合.最终将这5条gpl20序列都克隆人真核表达载体并表达出糖基化的gp120蛋白.结论 在设计疫苗和检测试剂时应考虑到膜区的高变性,gp120糖蛋白的体外表达有利于进一步开展针对我国主要HIV流行毒株膜蛋白致病机制和疫苗学的研究.
目的 分析我國HIV-1主要亞型毒株的gp120區域的序列特徵,併錶達齣gp120糖基化蛋白,為研究gp120的緻病機製和設計疫苗奠定基礎.方法 利用巢式PCR從來自于不同省份的5名HIV-1感染者外週血單箇覈細胞DNA中擴增全長gp120基因併測序,對序列特徵進行分析,併將穫得的gp120基因剋隆入真覈錶達載體,體外錶達穫得gp120糖基化蛋白.結果 序列分析顯示,此5條gp120序列分屬HIV-1 Thai-B、BC重組和AE重組啞型,雖然亞型不同,但這些gp120序列在相同的位置都存在著一些保守的糖基化位點,且都具備相同的Furin蛋白酶第一切割位點,與參攷序列比較髮現,不同的HIV亞型序列中,除V3序列長度較為保守外,V1、V2、V4、V5各區域都有不同程度的缺失現象,與BC重組和AE重組亞型相比,Thai-B亞型V3的頂耑環呈現齣多種組閤.最終將這5條gpl20序列都剋隆人真覈錶達載體併錶達齣糖基化的gp120蛋白.結論 在設計疫苗和檢測試劑時應攷慮到膜區的高變性,gp120糖蛋白的體外錶達有利于進一步開展針對我國主要HIV流行毒株膜蛋白緻病機製和疫苗學的研究.
목적 분석아국HIV-1주요아형독주적gp120구역적서렬특정,병표체출gp120당기화단백,위연구gp120적치병궤제화설계역묘전정기출.방법 이용소식PCR종래자우불동성빈적5명HIV-1감염자외주혈단개핵세포DNA중확증전장gp120기인병측서,대서렬특정진행분석,병장획득적gp120기인극륭입진핵표체재체,체외표체획득gp120당기화단백.결과 서렬분석현시,차5조gp120서렬분속HIV-1 Thai-B、BC중조화AE중조아형,수연아형불동,단저사gp120서렬재상동적위치도존재착일사보수적당기화위점,차도구비상동적Furin단백매제일절할위점,여삼고서렬비교발현,불동적HIV아형서렬중,제V3서렬장도교위보수외,V1、V2、V4、V5각구역도유불동정도적결실현상,여BC중조화AE중조아형상비,Thai-B아형V3적정단배정현출다충조합.최종장저5조gpl20서렬도극륭인진핵표체재체병표체출당기화적gp120단백.결론 재설계역묘화검측시제시응고필도막구적고변성,gp120당단백적체외표체유리우진일보개전침대아국주요HIV류행독주막단백치병궤제화역묘학적연구.
Objective To characterize 5 gpl20 sequences from mainly circulating clades in China and expression of their gp120 glycoproteins. Methods gp120 genes were amplified from the PBMCs of 5 HIV-1 infected individuals in different provinces using nest PCR and their DNA sequences were determined. Sequence characteristics were analyzed and gp120 genes were sub-cloned into the mammalian expression vec-tor to produce gp120 glycoproteins. Results Sequence characteristics indicated these sequences belong to the clade Thai-B, CRF_BC and CRF_AE, respectively. There were some conservative N-linked glycosyla-tion sites and primary Furin protease cleavage motifs in the same positions within gp120 amino acid se-quences although these gp120 sequences were categorized into different clades. In comparison with referen-tial strains, amino acids deletions were found in the V1, V2, V4, V5 regions except for the V3 loop; above all, V3 tip motifs of Thai-B exhibited the more polymorphic forms than those of CRF_BC and CRF_AE. These 5 gp120 sequences were cloned into the eukaryotic expression vector and gpl20 glycoproteins were produced successfully. Conclusion Hyper-variable nature of Env should be considered while designing HIV-1 vaccine or test reagent, and gpl20 expression in vitro is helpful to further research on the Env patho-genesis and vaccine development against the mainly circulating HIV-1 isolates in China.
