乙型肝炎病毒B，C基因型X蛋白在原核系统中的克隆表达与纯化
发布时间：2018-12-12 05:04
【摘要】： 目的:应用分子克隆技术获得乙型肝炎病毒(HBV)B、C基因型X蛋白,为进一步研究X基因在乙型肝炎病毒所致疾病中的作用奠定基础。 方法: ①基因克隆:应用PCR技术,分别从本实验室构建的B、C基因型HBV保守的X基因真核表达重组子—pcDNA3.1-Xb和pcDNA3.1-Xc中获取X全长基因,产物经EcoR I和XhoI酶切,分别与原核表达载体pET-42a连接,转化BL-21感受态细胞,抗生素筛选阳性重组子,PCR、酶切、测序鉴定。 ②蛋白表达纯化:取鉴定后的B,C基因型X基因重组菌,IPTG诱导重组蛋白的表达,SDS-PAGE电泳,用GST抗体经Western Breeze化学发光法鉴定融合蛋白。优化诱导时间和IPTG浓度,诱导重组蛋白大量表达;低温诱导可溶性X蛋白的形成。分别用GST和Ni-NTA bind纯化试剂盒纯化X蛋白,比较两种方法的纯化效果,并用Bradford法测定重组蛋白浓度。 结果: ①PCR法获取的乙型肝炎病毒B、C基因型X基因的大小与预期一致;克隆后筛选的阳性重组子经PCR、酶切、测序鉴定正确。 ②重组子均可被IPTG诱导表达融合蛋白,表达产物可被GST抗体识别;当诱导时间为2 h、IPTG终浓度为0.5 mMol /L时,表达量均达最大,约为菌体总蛋白的40%以上;18℃,25℃,30℃均难以诱导可溶性蛋白的产生;溶解后的包涵体经复性后进行GST纯化,但是最终未能得到目的蛋白;而用Ni-NTA纯化后可直接得到大量融合蛋白,而且纯度可达95%以上。 结论:成功构建了B、C基因型乙型肝炎病毒X基因的原核表达系统,并获得高效表达;重组蛋白经Ni-柱纯化后可得到大量重组蛋白,且纯度较高,为进一步研究X基因在乙型肝炎病毒所致疾病中的作用奠定基础。
[Abstract]:Objective: to obtain the X protein of hepatitis B virus (HBV) B C genotype by molecular cloning technique, and to lay a foundation for further study on the role of X gene in hepatitis B virus induced diseases. Methods: 1 Gene cloning: using PCR technique, X full-length genes were obtained from pcDNA3.1-Xb and pcDNA3.1-Xc, the eukaryotic expression recombination of X gene conserved by HBV, which was constructed in our laboratory. The product was digested by EcoR I and XhoI, ligated with prokaryotic expression vector pET-42a, transformed into BL-21 receptive cells, screened for antibiotic positive recombinant, digested by PCR, and identified by sequencing. 2 protein expression and purification: the recombinant strain of X gene was obtained and the recombinant protein was induced by IPTG. SDS-PAGE electrophoresis was used to identify the fusion protein by Western Breeze chemiluminescence assay with GST antibody. The induction time and concentration of IPTG were optimized to induce the expression of recombinant protein and the formation of soluble X protein was induced by low temperature. X protein was purified by GST and Ni-NTA bind purification kit respectively. The purification effect of the two methods was compared and the concentration of recombinant protein was determined by Bradford method. Results: the size of genotype X gene of hepatitis B virus (HBV) obtained by 1PCR method was the same as expected, and the positive recombinant was digested by PCR, and sequenced. 2Recombinant could be induced by IPTG to express the fusion protein, and the expression product could be recognized by GST antibody, and when the induction time was 2 h, the final concentration of IPTG was 0.5 mMol / L, the expression reached the highest level, which was more than 40% of the total bacterial protein. It was difficult to induce the production of soluble protein at 18 鈩，
本文编号：2373961
[Abstract]:Objective: to obtain the X protein of hepatitis B virus (HBV) B C genotype by molecular cloning technique, and to lay a foundation for further study on the role of X gene in hepatitis B virus induced diseases. Methods: 1 Gene cloning: using PCR technique, X full-length genes were obtained from pcDNA3.1-Xb and pcDNA3.1-Xc, the eukaryotic expression recombination of X gene conserved by HBV, which was constructed in our laboratory. The product was digested by EcoR I and XhoI, ligated with prokaryotic expression vector pET-42a, transformed into BL-21 receptive cells, screened for antibiotic positive recombinant, digested by PCR, and identified by sequencing. 2 protein expression and purification: the recombinant strain of X gene was obtained and the recombinant protein was induced by IPTG. SDS-PAGE electrophoresis was used to identify the fusion protein by Western Breeze chemiluminescence assay with GST antibody. The induction time and concentration of IPTG were optimized to induce the expression of recombinant protein and the formation of soluble X protein was induced by low temperature. X protein was purified by GST and Ni-NTA bind purification kit respectively. The purification effect of the two methods was compared and the concentration of recombinant protein was determined by Bradford method. Results: the size of genotype X gene of hepatitis B virus (HBV) obtained by 1PCR method was the same as expected, and the positive recombinant was digested by PCR, and sequenced. 2Recombinant could be induced by IPTG to express the fusion protein, and the expression product could be recognized by GST antibody, and when the induction time was 2 h, the final concentration of IPTG was 0.5 mMol / L, the expression reached the highest level, which was more than 40% of the total bacterial protein. It was difficult to induce the production of soluble protein at 18 鈩，
本文编号：2373961
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2373961.html
最近更新
教材专著
