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

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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