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通过PCR人工合成模板的方法获得牛Tα1基因,与含IFNα-2b基因的pAG-IFN重组质粒,构建Tα1/IFNα-2b融合基因重组质粒pUC18-Tα1/IFN,经序列分析证实,融合基因Tα1和IFNα-2b与GenBank登录基因的序列一致性分别为100%和98.5%,仅IFNα-2b有两处碱基发生无义突变.再将融合基因亚克隆入pBV220,构建融合表达载体pBV220-Tα1/IFN,转化到E.coliM15经IPTG诱导,实现了Tα1-IFNα-2b融合蛋白的高表达,约占菌体总蛋白的24.5%,为包涵体形式.这为研制Tα1-IFNα-2b双重活性的融合蛋白奠定了基础.
The recombinant plasmid pUC18-Tα1 / IFN of Tα1 / IFNα-2b fusion gene was constructed by PCR artificial synthesis of the template and the pAG-IFN recombinant plasmid containing IFNα-2b gene. The sequence analysis confirmed that the fusion gene Tα1 and The sequence identities of IFNα-2b and GenBank accession genes were 100% and 98.5%, respectively, and only two non-sense mutations of IFNα-2b were found in IFNα-2b.The subcloned fusion gene was subcloned into pBV220 to construct the fusion expression vector pBV220-Tα1 / IFN and transformed into E.coli M15 induced by IPTG, which resulted in the high expression of Tα1-IFNα-2b fusion protein, accounting for 24.5% of the total bacterial protein, which was in the form of inclusion body. Fusion protein laid the foundation.