肝癌相关基因HTA的重组表达及多克隆抗体的制备

Recombinant expression of Hepatoma Associated Gene HTA and preparation of anti-HTA polvclonal antibodv

目的将肝癌相关基因HTA进行克隆、表达并制备多克隆抗体,为进一步研究其作为肝癌导向治疗靶点的可行性及临床意义奠定基础。方法应用RT-PCR技术,从人肝癌细胞系HepG2细胞中扩增得到HTA_(338-616)cDNA,再将其克隆到原核表达载体pET21a(+)-MBP内,用IPTG诱导其在大肠杆菌BL21(DE3)中表达。His-tag磁珠纯化试剂盒纯化重组蛋白MBP-HTA,通过Western blot和ELISA方法检测重组蛋白的抗原性。将纯化的重组蛋白免疫BALB/c小鼠制备多克隆抗体,并采用ELISA、Western blot和免疫组化的方法检测抗体的灵敏度和特异性。结果成功地构建了表达MBP-HTA融合蛋白的原核表达质粒pET21a(+)-MBP-HTA。重组MBP-HTA融合蛋白在大肠杆菌BL21(DE3)内得以高效表达,且以包涵体的形式存在。His-tag磁珠纯化试剂盒纯化和Western blot分析,得到了分子量约为52kDa的目的蛋白。获得了高效价的特异性多克隆抗体,经ELISA检测抗体的效价为1:3200,用Western blot检测的效价为1:400。免疫组织化学检测表明:肝癌组织中HTA蛋白的阳性表达率明显高于正常肝组织(P<0.01)。结论成功地制备出抗MBP-HTA多克隆抗体,该抗体有较高的效价和特异性;能用于免疫组织化学的检测,且HTA在肝癌组织中的阳性表达率明显高于正常肝组织HTA蛋白有望成为肝癌导向治疗的潜在靶点。

Objective Recombinant expression of Hepatoma Associated Gene（I-ITA） and prepm-ation of antiTA polyclonal antibody for the further study as an oriented therapeutic target of hepatocellular carcinoma and clinical significance of the feasibility of the basis. Method HTA338-616 was amplified from HepG2 cells and cloned into the prokaryotic expression vector pET21a（＋）-MBP, then expressed in E.coli BL21（DE3） which was induced by WFG. The recombinant protein MBP-HTA was purified by His-tag magnetic bead purification kit and identified by Western blot and ELISA method. Polyclonal antibodies was developed by immunizing BALB/c mice with purified recombinant protein, and their sensitivity and specificity were tested using ELISA,Westem blot and immunohistochemistry staining analysis. Results The prokaryotic expression plasmid pET21a（＋）-MBP-HTA was successfully constructed. The recombinant protein MBP-HTA could be expressed in abundance in the form of inclusion bodies. We got the purified purpose protein of 52kDa and Westem blot analysis showed that the His-tag was expressed correctly. High titer polyclonal antibody with a high specificity was obtained. Using ELISA and Western blot analysis, theliter of the polyclonal antibody was 1 ： 3200 and 1 ： 400 respectively. Compared with the normal hepatic tissues, the positive expression rate of HTA pmlein was obviously higher in hepatocellular carcinoma tissue （P〈0.01）. Conclusion The polyclonal antibody against MBP-HTA with high sensitivity and high specificity was successfully prepared, and can be applied in immunohistochemistry staining with the result of obviously higher positive expression rate of HTA protein in hepatocellular carcinoma tissues than in normal hepatic tissues. HTA protein may be a potential oriented therapeutic target of hepatocellular carcinoma.