【摘要】 目的:克隆猪的BCL-GL基因,进行原核表达,并制备出其多克隆抗体。方法:以提交NCBI的人的BCL-GL基因序列为种子序列,对猪的ESTs数据库进行比对拼接,得到contig序列。根据这个序列设计克隆引物,以猪脾脏总RNA反转录得到的cDNA为模板,PCR得到克隆序列。克隆序列经测序验证后,连接到原核表达载体pET-32a,构建成重组表达载体pET32a-BCL-GL,然后将重组载体转化到大肠杆菌BL21进行诱导表达。经His-标签融合蛋白纯化试剂盒纯化得到纯化蛋白制备豚鼠多克隆抗体。结果:抗体ELISA效价为1∶800,Western blot反应特异性良好。结论:成功地克隆了猪BCL-GL基因,并进行了原核表达,制备了特异性豚鼠抗BCL-G血清,为进一步研究其功能奠定基础。更多还原

【Abstract】 AIM: In order to express a novel gene named as BCL-GL of swine in E.coli and prepare its polyclonal antibody.METHODS: The contig sequence of the gene was predicted and in silicon cloned by blasting the human BCL-GL in swine ESTs database in NCBI.The cloning sequence was obtained by RT-PCR from swine spleen.The cloning sequence was identified by sequencing and compared with the contig sequence.Then the gene was cloned into a prokaryotic expression vector pET-32a to construct a recombinant plasmid named as pET32a-BCL-GL.The fusion protein pET32a-BCL-GL was expressed in E.coli BL21 and purified using a His-tag fusion protein purification kit.Then guinea pigs were immunized with the purified protein to get the specific polyclonal antibody.RESULTS: The titer of the antibody was 1∶800 detected by ELISA.The protein BCL-GL can be specifically detected by western blot assay using the polyclonal antibody.CONCLUSION: The novel swine gene BCL-GL was cloned and expressed in E.coli and its polyclonal antibody was prepared successfully.更多还原