摘要
目的:制备HER-2抗原疫苗.方法:采用RT-PCR方法从HER-2+SKBR3细胞株中获得HER-2基因胞外段编码区cDNA,构建pQE-30-HER-2原核表达载体,在大肠杆菌中表达HER-2胞外区蛋白.Ni-NTA柱亲和层析纯化后,梯度尿素复性,Western Blot鉴定.用获得的蛋白免疫小鼠,检测重组蛋白在小鼠体内诱导的免疫应答.结果:构建的pQE-30-HER-2质粒经酶切和DNA测序分析,结果表明该基因的序列与设计的序列完全相同.构建的质粒转化DH5α后经IPTG诱导表达,SDS-PAGE分析,该重组蛋白以包涵体形式表达,且与mAb Herceptin有较好的结合活性.包涵体用Ni-NTA柱亲和层析纯化后,梯度尿素复性,最大限度的获得了可溶性的HER-2胞外区蛋白.用重组蛋白免疫小鼠,得到了较高的抗体滴度.结论:成功制备了HER-2蛋白疫苗并诱导出较高抗体滴度,为下一步工作打下基础.
Abstract
AIM: To prepare the vaccine of HER-2 antigen. METHODS: cDNA of HER2 extracellular domain was prepared through RT-PCR from HER-2 + SKBR cell line, cloned into the multiple cloning site of pQE-30 and expressed in E. coli DH5α. The expressed protein was purified through Ni-NTA resin affinity chromatography. Eluted protein was refolded by different concentrations of urea and then detected by Western Blotting. The protein immunization antibody detection in immunized mice was examined by ELISA. RESULTS: DNA sequencing and restriction enzyme digestion proved that HER-2 gene was correctly connected. SDS-PAGE and Western blotting analysis showed that the protein was expressed in conclusion body in E. coli DH5α and bound to Herceptin. We purified the recombinant protein by Ni-NAT affinity chromatography and obtained soluble human HER-2 extracellular domain. Immune response was effectively induced by rhHER-2. CONCLUSION: HER-2 protein vaccine has been successfully prepared and effective immune response has been induced.
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维普
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