抗preS1分子高效单克隆抗体的制备及其在血清LHBs检测中的应用  

Preparation and identification of monoclonal antibody against preS1 and its application in detection for HBV large surface protein

作　　者：李新军[1] 张竹君[1] 易维京[1] 陈安[1] 李淑慧[1] 赵忠颢[1] 刘明栋 范毓东[3] 胡川闽[1] 

机构地区：[1]第三军医大学:医学检验系临床生物化学教研室,重庆400038 [2]西南医院全军感染病研究所,重庆400038 [3]西南医院肝胆外科,重庆400038

出　　处：《第三军医大学学报》2011年第6期566-569,共4页Journal of Third Military Medical University

基　　金：国家科技重大专项课题(2009ZX10607);第三军医大学科技成果转化基金课题(2009XZH03)~~

摘　　要：目的制备可用于血清LHBs检测的针对preS1高效单克隆抗体并进行初步临床应用研究。方法对乙肝preS1区段基因（B基因型,adr血清亚型）全长序列（1～119 aa）进行密码子偏嗜性改造后,克隆入质粒pET32a构建原核表达重组质粒并转化入大肠杆菌BL21（DE3）,采用离子交换、HIS亲和层析柱以及肠激酶（EK酶）酶切等方法建立preS1重组蛋白的纯化工艺,制备筛选单克隆抗体的筛选原;综合应用多种生物信息学分析软件预测LHBs preS1区段B细胞表位肽preS1（36～58 aa）,合成该肽段后与BSA交联,通过新型免疫程序制备单克隆抗体;建立以抗preS1高效单克隆抗体为基础的ELISA方法,并对临床血清LHBs进行检测分析。结果获得了高纯度的重组preS1蛋白,HPLC分析其纯度为98%,通过新型免疫程序制备的抗preS1单克隆抗体1-E6（IgG1）特异性好,对preS1（LHBs）的检出率（83.3%）高于其他同类抗体（P〈0.01）。结论获得高效特异的抗preS1单克隆抗体1-E6以及高纯度重组preS1蛋白免疫检测标准品。Objective To prepare monoclonal antibodies（mAbs） against preS1 to detect large surface protein（LHBs） in the serum of HBV carriers and investigate its clinical value.Methods According to the frequency of E.coli codons,an E.coli favorite DNA fragment,encoding amino acids sequence identical to that of the wild type preS1（1 to 119 aa）（genetypeB,serotype adr） was cloned into plasmid pET32a.The preS1 protein was induced to express and then purified by cation exchange,His-tag affinity column,and then digested by enterokinase before it was used to screen its monoclonal antibody.LHBs preS1 B cell epitope peptide（36 to 58 aa） was forecast with aid of bioinformatics analysis softwares.Then the peptide was synthesized and then cross-linked with BSA.BALB/c mice were immunized with this cross-linked peptide through a new immunization procedure to produce mAb against preS1.An ELISA method was established to detect LHBs in the serum with these mAbs in blood serum samples of 413 identified hepatitis patients and 100 health individuals,and the results were compared with those of other commercial detection method.Results Highly purified preS1（HPLC indicated a purity of 98%） was obtained.Our renewed immunization generated mAb（1-E6）（IgG1 subclass） against preS1 with good specificity.The positive rate of preS1 was higher when our mAbs 1-E6 was used than other commercial mAbs was in detection of LHBs with ELISA（83.3% vs 72.9%,P0.01）.Conclusion We obtain preS1 protein with high purity and suitable as standard substance,and efficient and specific mAb（1-E6） in detection of LHBs with ELISA.

关 键 词：PRES1 单克隆抗体 LHBs检测 ELISA 

分 类 号：R392-33[医药卫生—免疫学] R392.11[医药卫生—基础医学]