人绒毛膜促性腺激素β亚基单一B-细胞表位(β5、β9或β8)融合蛋白的表达和纯化

Expression and Purification of Three Fusion Proteins Containing a Single B|cell Epitope (β5、β9 or β8) of Human Chorionic Gonadotropin β Subunit

作为开发新型实用性人绒毛膜促性腺激素 (hCG)疫苗的一种尝试 ,我们已构建若干组合靶抗原三个线性B_细胞表位和外源强T_细胞表位的基因工程hCG嵌合肽。为了检测用这些嵌合肽免疫的动物血清中是否能产生抗各表位的三种抗体 ,本研究选用能在大肠杆菌中高表达和与生物素亲和性强且特异 (方便通过亲和层析纯化 )的链霉亲和素为载体 ,分别构建了三种含 β_hCG不同单一线性B_细胞表位 (β5 ,β9和 β8)的融合蛋白。在链霉亲和素基因下游多克隆区EcoRⅠ和HindⅢ位点插入各表位编码基因片段 (带TAA终止密码子 )的pTSA_18重组质粒 ,转化BL2 1(DE3)pLysS宿主菌后 ,它们在IPTG诱导下均能以较高水平表达各自目的融合蛋白 ,而且它们的表达产物在Westernblot鉴定中都能被抗各表位特异的多抗或单抗或抗报告表位单抗识别。用改良的制备性PAGE方法可以一步纯化电泳均一性高于 95 %的三个融合蛋白 ,它们的收得率相对 1L培养物约为 5mg。作为化学合成表位肽的替代物 ,β_hCG三个单一B_细胞表位融合蛋白的可获得性将有助于所构建hCG基因工程嵌合肽以及其他hCG疫苗 ,也包括它的DNA疫苗的免疫原性分析。

The possibility of using a subunit or fragment of human chorionic gonadotropin (hCG) as an immunogen for birth control has been actively explored for many years. This protein homone is produced by the fertilized egg and is required for implantation of the blastocyst into the maternal uterus and the maitenance of pregnancy. In previous studies, several bio_synthesized hCG chimeric peptides (CP) that contain three linear B_ cell epitopes (β5, β9 and β8) of β_hCG subunit together with various foreign `promiscuous' T_ cell epitopes were constructed and expressed as potential new hCG vaccine immunogens. In order to detect antibodies to each of the individual B_cell epitopes present in the animal antiserum raised against the hCG CPs, we decided to construct three recombinant proteins, each contains a single target B_ cell epitope (βE) of β_hCG. Two sets of DNA fragments were chemically synthesized encoding the β5, β9 and β8 epitopes (βE) 45～52, 113～116 or 133～144 of β_hCG subunit and were inserted into the downstream of streptavidin (Stv) gene in pTSA18 separately, with or without an extra TAA codon at the 3'_ terminals of the genes. SDS_PAGE analysis revealed that only Stv_βE (-β5, -β9 or -β8) fusion genes set with the TAA codon can be expressed in E.coli BL21(DE3)pLysS strain at high level after 1mM IPTG induction for 4 hours. Additionally, these fusion proteins can all be recognized by specific polyclonal antiserum (RS_4157) generated upon immunization with the loop peptide 38～57 of β_hCG, monoclonal antibody (mAb) FB12 to β9 epitope and mAb OT3A that specially recognizes reporter sequence 133～139 of β8 epitope 137～144. Each of the proteins can be purified to 95% relative homogeneity using an improved method of preparative gel polyacrylamide gel electrophoresis. The yields were 5 mg per 1L culture. The three target Stv_βE fusion proteins will be useful in determining the immunogenicity of designed hCG CPs and hCG vaccines, including hCG DNA vaccines.