hK7多克隆抗体的制备及其在前列腺中的初步应用  

Anti-hK7 Polyclonal Antibody Generation and Preliminary Investigation of hK7 Expression in Prostate

作　　者：黄逢雨[1] 莫曾南[2] 

机构地区：[1]烟台市莱阳中心医院泌尿外科,烟台265200 [2]广西医科大学第一附属医院泌尿科学研究所,南宁530021

出　　处：《中国医药导刊》2013年第6期1089-1091,1093,共4页Chinese Journal of Medicinal Guide

摘　　要：目的:构建KLK7原核表达载体,诱导hK7蛋白表达,制备抗hK7多克隆抗体。并利用此抗体研究hK7在正常前列腺,前列腺增生和前列腺癌三种组织中表达的差异及其意义。方法:PCR扩增KLK7基因,和pET-21a载体构建pET-21a-KLK7重组表达质粒。转化大肠杆菌,筛选阳性克隆,IPTG诱导表达,纯化融合蛋白并鉴定,免疫家兔获得抗血清,ELISA检测抗体的效价。免疫组化检测组织芯片中正常前列腺,前列腺增生及前列腺癌组织中hK7蛋白的表达情况。结果:成功构建了pET-21a-KLK7重组表达质粒,经转化、诱导表达和纯化,SDS-PAGE证实重组hK7蛋白与预期结果一致。免疫家兔,得到抗血清,ELISA检测抗体效价为1:200万以上。免疫组化显示在前列腺癌中的表达要比在正常前列腺及前列腺增生中的表达下降(P<0.05)。结论:获得了高纯度的hK7蛋白,及高效价的多克隆抗体,前列腺癌中hK7的表达较正常和增生前列腺组织降低,hK7可能有潜在抑制前列腺癌的作用。Objective：To construct the recombinant vector,induce expression of the recombinant protein,and obtain rabbit anti-hK7 polycolonal antibody.To investigate the expression difference of human tissue Kallikreins 7（hK7）in normal prostate、BPH and prostate carcinoma.Methods：KLK7 gene was amplified by PCR.And the pET-21a-KLK7 recombinant plasmid was constructed and amplified by transformation into competent E.coli BL21（DE3）.Colony PCR was used to screen and identify positive clone.The expression of HishK7 fusion protein was induced with IPTG.The production was purified twice and analyzed by SDS-PAGE.The antiserum was obtained by immunizing rabbits with the purified hK7 protein.The titre of antibody was detected by ELISA.The immunohistochemical assay was used to detect the expression of hK7 in Tissue Array with normal prostate tissue 18 dots,BPH 49 dots,and prostate carcinoma 26 dots.Results：The pET-21a-KLK7 recombinant plasmid was constructed successfully.After the recombinant plasmid was transformed into E.coli BL21（DE3）,His-hK7 fusion protein induced with IPTG.Purified protein was detected near 26KD by SDS-PAGE.The antiserum was obtained by immunizing 2 rabbits with the purified hK7 protein.The titer of the antibody in the rabbit serum was greater than 2,000,000.The positive expression of hK7 in prostate carcinoma was significant lower than that in normal prostate and in BPH.Conclusion：The high-purity recombinant protein hK7 and high titer antibody was successfully prepared,which will lay the foundation for the detection and research of hK7.The expression of hK7 in prostate carcinoma is lower significantly than that in normal prostate and BPH.The downregulation of hK7 in prostate carcinoma may point out to be a possible inhibitory effect of the gene on prostatic tumar growth.

关 键 词：人类组织激肽释放酶7 多克隆抗体 前列腺增生 前列腺癌 组织芯片 

分 类 号：R737.25[医药卫生—肿瘤] R73-3[医药卫生—临床医学]