HIV-1整合酶C末端及N末端结构域共结合多肽候选药物的筛选  

Screening of HIV-1 integrase CTD and NTD co-binding candidate peptide drugs

作　　者：叶丽颖 彭臻菲[1] Liying Ye;Zhenfei Peng(Fujian Health College,Fuzhou 350101,China)

机构地区：[1]福建卫生职业技术学院,福州350101

出　　处：《中华细胞与干细胞杂志（电子版）》2022年第5期257-265,共9页Chinese Journal of Cell and Stem Cell（Electronic Edition）

摘　　要：目的利用双分子荧光互补技术筛选人类免疫缺陷病毒1(HIV-1)的整合酶(IN)C末端结构域(CTD)及N末端结构域(NTD)多肽类药物。方法构建用于双分子荧光互补技术筛选的HIV-1整合酶CTD及NTD结构域的重组质粒pBiFc-VN173-Flag-IN-CTD及pBiFc-VN173-Flag-IN-NTD,并进行限制性内切酶酶切及测序鉴定。将重组质粒pBiFc-VN173-Flag-IN-CTD、pBiFc-VN173-Flag-IN-NTD及对照质粒pBiFC-VN173-Flag分别转染HEK293T细胞,使用蛋白免疫印迹法和Flag抗体检测2种重组质粒的表达。将3组质粒分别与多肽药物表达文库pBiFc-VC155-TrxA-11AA-TrxA共转染HEK293T细胞,在荧光显微镜下观察绿色荧光强度。将筛选到的多肽表达载体pcDNA-cmyc-11AA及对照pcDNA-cmyc分别转染HEK293T细胞,24 h后再用表达绿色荧光蛋白的慢病毒感染,36 h后观察绿色荧光强度。多组间比较用单因素方差分析,多个组与同一对照组比较采用Dunnett-t检验。结果经酶切及测序结果确认,重组质粒pBiFc-VN173-Flag-IN-CTD及pBiFc-VN173-Flag-IN-NTD构建成功,用Flag抗体可检测到两种质粒表达的重组蛋白。多肽文库中peptide 4和peptide 192与重组质粒分别共转染HEK293T细胞后,在荧光显微镜下可观察到绿色荧光的出现。经慢病毒感染后,与对照比较,转染多肽表达载体pcDNA-cmyc-peptide4和pcDNA-cmyc-peptide192的HEK293T细胞中荧光细胞数[(78.33±5.81)、(29.33±2.96)比(350.30±8.67)个]减少,差异有统计学意义(P<0.05)。结论用于双分子荧光互补技术筛选的HIV-1整合酶CTD及NTD表达载体构建成功,并筛选获得靶向2个结构域的多肽peptide 4和peptide 192,其可能通过和整合酶的结合而抑制慢病毒的整合。Objective To screen human immunodeficiency virus 1(HIV-1)integrase(IN)C-terminal domain(CTD)and N-terminal domain(NTD)peptide drugs using bimolecular fluorescence complementation(BiFC)technology.Methods The recombinant plasmids pBiFc-VN173-Flag-IN-CTD and pBiFc-VN173-Flag-IN-NTD of HIV-1 integrase CTD and NTD were constructed for the screening of HIV-1 integrase by BiFC technology and identified by restriction endonuclease enzyme and sequencing.The recombinant plasmids(pBiFc-VN173-Flag-IN-CTD and pBiFc-VN173-Flag-IN-NTD)and the control plasmid pBiFC-VN173-Flag were transfected into HEK293T cells,respectively,and the expression levels of fused proteins were identified by Western blotting with anti-Flag antibody.The two recombinant plasmids and the control plasmid were co-transfected into HEK293T cells with the peptide library pBiFc-VC155-TrxA-11AA-TrxA,respectively,then the intensity of green fluorescence was observed under a fluorescence microscope.The selected polypeptide expression vector pcDNA3.1-cmyc-11AA and the control vector pcDNA3.1-cmyc were transfected into HEK293T cells,then after 24 hours the cells were infected with lentivirus expressing green fluorescence protein,and the green fluorescence intensity was observed 36 hours later.Results The recombinant plasmids pBiFc-VN173-Flag-IN-CTD and pBiFc-VN173-Flag-IN-NTD were confirmed by restriction endonuclease enzyme digestion and sequencing,and the fused proteins expressed by these two plasmids could be detected with anti-Flag antibody.In addition,peptide 4 and peptide 192 were screened from the peptide library after co-transfection with recombinant plasmids in HEK293T cells based on the occurrence of green fluorescence.After lentivirus infection,the fluorescence intensity of HEK293T cells transfected with peptide expression vectors pcDNA3.1-cmyc-peptide 4 and pcDNA3.1-cmyc-peptide 192 was significantly decreased compared with the control.Conclusions Two plasmids expressing Flag-fused HIV-1 integrase CTD and NTD were successfully constructed and confirmed.

关 键 词：HIV-1整合酶 NTD CTD 多肽类药物 双分子荧光互补技术 

分 类 号：R943[医药卫生—药剂学]