抗人免疫缺陷病毒I型(HIV-1)外膜蛋白gp120单链抗体基因的构建及测序鉴定  

Construction and Sequence Analysis of the Single-chain Fv Gene of Specific for Anti-HIV-1 gp120

作　　者：许铭炎[1] 洪坤学[2] 邓小玲[1] 于晓军[1] 徐小虎[1] 陈梅珍[1] 邵一鸣 

机构地区：[1]汕头大学医学院病原生物学教研室,广东汕头515041 [2]中国疾病控制中心性病艾滋病中心病毒免疫室,北京100050

出　　处：《江西医学院学报》2005年第1期14-16,27,共4页Acta Academiae Medicinae Jiangxi

基　　金：国家自然科学基金课题(NO.30070291)

摘　　要：目的　构建抗HIV 1gp120单链抗体基因,为进一步用于HIV 1感染的诊断和治疗奠定基础。方法　利用 RT PCR法,从抗HIV 1gp120单克隆抗体杂交瘤细胞扩增得到抗体轻链和重链的可变区基因,经重叠延伸反应, 在体外随机合成单链抗体基因(ScFv),并克隆到pGEM Easy T载体中,经测序及Blast同源性分析。结果　该 ScFV基因全长为666bp,为VH Linker VL结构,VH基因为396bp,编码132个氨基酸;Linker为(Gly4Ser)3短 肽;VL基因为270bp,编码90个氨基酸。VH基因与小鼠IgG2a重链可变区的同源性达95%,VL基因与小鼠免 疫球蛋白κ轻链可变区的同源性达98%。结论　成功构建了抗HIV 1gp120单链抗体基因。Objective To construct the single chain variable fragment (ScFv) gene specific for anti-HIV-1gp120 that could be used in antibody-directed diagnosis and therapy for HIV-1 infection.Methods The heavy-chain and light-chain variable region gene of immunoglobulin were amplified individually from the anti-HIV-1gp120 monoclonal hybridoma cell mRNA by RT-PCR and joined by a DNA linker encoding peptide (Gly 4Ser) 3 with overlap extension,as a single-chain Fv (ScFv) DNA fragment.The ScFv fragment was cloned into the vector pGEM-Easy-T,sequenced with auto-DNA sequencer and analyzed by Nucleotide Blast in NCBI.Results The ScFv gene was the structure of VH-Linker-VL,consisting of 666 bp.The VH gene was 396bp,encoding 132 amino acids.The sequence of the linker was (Gly 4Ser) 3.The VL gene was 270bp encoding 90 amino acids.The identity of VH gene and mouseγ2a heavy-chain variable region of immunoglobulin gene was 95%.The identity between VL gene and mouseκlight-chain variable region of immunoglobulin gene was 98%.Conclusion The gene of anti-HIV-1gp120 ScFv is constructed.

关 键 词：单链抗体 基因克隆 测序 人免疫缺陷病毒1型 外膜蛋白gp120 

分 类 号：R392.11[医药卫生—免疫学]