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时间:2020-05-14
《抗人肝细胞生长因子受体单链抗体慢病毒载体的构建和应用.pdf》由会员上传分享,免费在线阅读,更多相关内容在行业资料-天天文库。
1、960细胞与分子免疫学杂志(ChinJCellMolImmuno1)2014,30(9·论著·文章编号:1007—8738(2014)09—0960—04抗人肝细胞生长因子受体单链抗体慢病毒载体的构建和应用陈永华,郭佳,尹衍新。,蒋明,朱红胜,张国栋,李冰宇(苏州市立医院东区检验科,同济大学苏州研究院生物医药中心,江苏苏州215000)[摘要]目的构建可表达抗人肝细胞生长因子受体(HGFR)单链抗体(seFv)的慢病毒表达载体,将其感染HEK293细胞,检测该抗体的表达及与抗原结合活性。方法采用反转录PCR(RT.PCR)从分泌小鼠抗人
2、HGFR抗体的杂交瘤细胞株(8E8)中扩增V和V.基因,用重叠延伸PCR法将V和V,进行拼接,得到含有信号肽sP-V一linker—V。.的单链抗体基因(HGFR-scFv),将其插入克隆载体pCR—Blunt中。用内切酶将HGFR—scFv基因从pCR-Blunt上切下,插入慢病毒载体pRRL—CMV中,经相应酶切和测序鉴定,正确构建慢病毒表达载体pRRLHGFR—scFv。磷酸钙法转染HEK293T细胞,48h后通过荧光显微镜检测绿色荧光蛋白(GFP)的表达,将获得病毒颗粒进一步感染HEK293细胞,RT·PCR和ELISA检测se
3、Fv的表达情况。结果抗人HGFR-scFv的慢病毒表达载体构建成功,病毒颗粒感染HEK293细胞后,得到了可以稳定表达抗人HGFR-scFv的细胞系;ELISA结果表明,scFv能与人HGFR特异性结合。结论成功克隆了小鼠抗人HGFR抗体的scFv基因,并构建其慢病毒表达系统。[关键词】肝细胞生长因子受体;单链抗体;慢病毒表达载体[中图分类号]R392.11,Q876,R446.61[文献标志码]AConstructionandapplicationofalentiviralvectorofsingle-chainvariablefra
4、gmentantibodyagainsthumanhepatocytegrowthfactorreceptorCHENYonghua,GUOJia,YINYanxin,JIANGMing。,ZHUHongsheng,ZHANGGuodong,LIBingyu。DepartmentofClinicalLaboratory,SuzhouMunicipalHospital,CenterforBio-Pharmacy,SuzhouInstitute,TongjiUniversity,Suzhou215000,ChinalAbstractJObj
5、ectiveToconstructalentiviralexpressionvectorcarryingthesingle—chainvariablefragment(scFv)antibodyagainsthumanhepatocytegrowthfactorreceptor(HGFR),expressitintransfectedHEK293cells,andobserveitsbiologicalfunctionofspecificbindingtoantigen.MethodsThevariableregionsofthehea
6、vychain(VH)andlightchain(VL)geneswereamplifieddirectlyfr0mthecDNAofhybridomacellline8E8secretingmouseanti-humanHGFRantibodyandassembledusingthespliceoverlapextension-PCR(SeE-PCR)。TheconstructedHGFR-scFvgenewiththesignalpeptideSP-VH"linker-VLwasligatedintothecloningvector
7、pCR-Blunt.Aftercutoffr0mpCR—Bluntusingenzymedigestion,HGFR—scFvgenewassubclonedintothelentiviraltransfervectorpRRL·CMV,whichwasidentifiedbytherestrictionenzymedigestionandsequencing.ThelentiviralexpressionvectorpRRLHGFR—scFvwasthentansfectedtogetherwiththepackagingplasmi
8、dsintoHEK293Tcelstoobtainvirusparticles,andgreenfluorescentprotein(GFP)expressionwasdetectedunderafluor
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