摘要
目的:获得人羧肽酶A基因,为进一步构建、表达抗人精浆蛋白单链抗体/羧肽酶A融合蛋白,用于前列腺癌的抗体导向酶-前体药物疗法奠定基础。方法:从人正常肺组织中提取总RNA,经反转录PCR分离人羧肽酶A基因,并克隆入pUC19质粒中,用全自动荧光测序仪测定其序列。将该目的基因插入pBV220载体,并转入大肠杆菌DH5α中,经热诱导表达重组蛋白。结果:获得了人羧肽酶A基因(由1251bp组成,可编码417个氨基酸),与国外发表的人羧肽酶AcDNA序列一致。表达的重组蛋白以SDSPAGE分析,在Mr为49000左右处出现1条新生蛋白带,表达量约占菌体总蛋白的17%。结论:成功地克隆人羧肽酶A基因,为构建表达抗人精浆蛋白/羧肽酶A的双功能抗体奠定了基础。
Aim :ToclonehumancarboxypeptidaseAgene,inorderforthegeneticconstructionandexpresstionof afusionproteinoftheScFvagainsthumanseminoproteinandcarboxypeptidaseA ,whichcouldbeusedinanti body -directedenzymeprodrugtherapyforprostatecancer.Methods:TotalRNAwasextractedfromhuman lungtissue ,andsubjectedtoreversetranscription.ThehumancarboxypeptidaseAgenewasamplifiedbyPCR , clonedintoplasmidvectorandanalysedbyuseofautoDNAsequencer .ThehumancarboxypeptidaseAgenewas insertedintoexpressionvectorpBV2 2 0andtransformedintoE .coliDH5α .Therecombinantproteinexpression wasinducedwith 4 2℃ .Results:HumancarboxypeptidaseAgeneconsistedof1 2 5 1bpencoding 4 1 7aminoacid residues,andwasthesameasthatreportedabroad .Afterinduction ,anewanticipatedproteinbandofMr 4 9 0 0 0appearedonSDS -PAGEgelandamountedaboutto 1 7%oftotalbacterialprotein.Conclusion :Thehuman carboxypeptidaseAgenewassuccessfullyclonedanditwouldbeusedtoconstructtheanti human seminoprotein/ carboxypeptidaseAbifunctionalantibody .
出处
《细胞与分子免疫学杂志》
CAS
CSCD
1999年第3期165-166,169,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
全军重点实验室基金!No.1 997 71 2 2
