论文部分内容阅读
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。
OBJECTIVE: To clone the BCL-GL gene of porcine for prokaryotic expression and to prepare its polyclonal antibody. Methods: The BCL-GL gene sequence of human NCBI was used as the seed sequence, and the pig ESTs databases were aligned and spliced to obtain the contig sequence. Based on this sequence, cloned primers were designed and cloned sequences were obtained by PCR using reverse transcribed total RNA of porcine spleen as template. The cloned sequence was verified by sequencing and ligated into the prokaryotic expression vector pET-32a to construct the recombinant expression vector pET32a-BCL-GL. The recombinant vector was transformed into E. coli BL21 for expression induction. The purified protein was purified by His-tag fusion protein purification kit to prepare guinea pig polyclonal antibody. Results: The titer of antibody was 1: 800 and the specificity of Western blot was good. CONCLUSION: The BCL-GL gene was successfully cloned and prokaryotic expressed. The specific anti-BCL-G serum was prepared for further study of its function.