通常情况下，外源蛋白的高效表达，与整合基因的拷贝数有关，符合基因剂量效应，故如何构建多拷贝载体，成为获得高表达重组菌株的重要手段。本文以内部核糖体结合位点(IRES)和自剪切2A多肽为工具，使外源基因以多顺反子的结构，在具有真核翻译后修饰和遗传稳定等特点的巴斯德毕赤酵母体系中进行表达，考察单、双基因拷贝数与蛋白表达之间的关系。研究对象为头孢菌素C(CPC)酰化酶，为生成头孢菌素类抗生素重要中间体-7-氨基头孢烷酸(7-ACA)的关键用酶，7-ACA的产量在医药销售市场中占有举足轻重的地位。本文所获得的研究成果如下:首先以pPIC3.5K穿梭质粒，构建含有单拷贝CPC酰化酶的重组质粒，然后利用IRES片段与2A多肽，以多顺反子的方式构建双拷贝CPC酰化酶的重组质粒，另外分别以IRES片段和2A多肽连接标记基因(GFP/hph)和CPC酰化酶基因的重组质粒，用来检测重组质粒上下游基因的表达水平。第二，以不同的酶切位点，将单拷贝质粒pPIC3.5K-CPCA进行线性化，使其以单交换(SacI)和双交换(BglII)的方式整合至酵母基因组中，而产生甲醇利用快速型(Mut+)和甲醇利用缓慢型(Muts)的重组菌株，其中Mut+表型的转化菌株最高酶活为1096U/L，其酶活几乎是Muts表型酶活的2倍，故后续实验皆以Mut+表型进行CPC酰化酶的表达。第三，将潮霉素B抗性基因(hph)连入IRES与2A序列的下游，利用潮霉素B抗性进行检测，最后于平板上得到具有潮霉素B抗性的重组菌株，以SDS-PADE进行分析，观察到hph蛋白的表达，上述结果都证实以IRES片段与2A多肽所构建的多顺反子结构在毕赤酵母中能够正常工作 。最后将双拷贝CPC酰化酶重组菌株，进行摇瓶发酵诱导，比较IRES片段和2A多肽重组菌株之间的CPC酰化酶的表达差异，实验表明IRES介导的重组菌株酶活为2751 U/L，而2A多肽的重组菌株最高酶活为2810.2 U/L。而与单拷贝子的最高酶活1096 U/L相比，酶活提高了1.6倍。上述工作，说明基因剂量与蛋白表达存在一定的正相关，为外源蛋白的高效表达提供了一条新的思路。
In general, highly effective expression of heterologous proteins has the connection with copy number of gene, accord with gene dosage effect. Nevertheless, a major problem of getting the high-expression recombinants is how to construct the multi-copy plasmid. In this research, we use the tools of internal ribosome entry site (IRES) and “self-cleaving” peptide 2A to construct the multicistronic structure. Then investigate the relationship between the copy numbers and the protein production in Pichia pastoris expression system that has the capability of performing many eukaryotic posttranslational modifications and the stability of expression strains.The object of study is cephalosporin C(CPC) acylase that can catalyze CPC by nonpolluting biomethod to produce 7-aminocephalosporanic acid (7-ACA), the most important intermediate of cephalosporins,and has achieved a dominant position in the medicine market.The research results are as follows:First, one-copy CPC acylase gene was introduced into the shuttle plasmid pPIC3.5K,also, double CPC acylase gene was introduced into IRES-mediated and 2A peptide-metidiated multicistronic structure as two-copy CPC acylase vector.in addition, a selective gene(GFP/hph) and CPC acylase gene was introduced into IRES-depedent and 2A peptide-dependent plasmid to test the gene expression level of upstream and downstream in the recombinant vector. Secondly, one-copy of recombinant vector was digested by SacI and BglII,which can integrated by signle-cross and double-cross into the Pichia pastoris genome,and,as a result, the transformant strains have two type phenotype, Mut+ and Muts. The optimal activity of Mut+ transformant reached 1096U/L, as much again the activity of Muts transformant.The result proved good conditions for the continued experiments.Thirdly,construction of IRES-dependent and 2A-dependent vector based on hygromycin B phosphotransferase gene for selection of hygromycin-resistant.The result show that the hygromycin-resistant transformant strain appeared on medium and the analysis of SDS-PAGE was also showed the protein production of hph.All result show the feasibility of constructing the IRES-mediated and 2A-mediated multicistronic plasimid in P. pastoris. After two-copy recombinants were induced in shake-flasks fermentation,compared the activity of CPC acylases between the IRES-dependent and 2A-dependent transformant. The data show that the optimal CPC acylase productivity of IRES- dependent transformant reached 2751U/L and 2A-dependent transformant reached 2810.2U/L, which activity is about 1.6 times the one-copy CPC acylase transformants. The above work can explain the positive correlations between copy number and protein expression and provide a new thinking for highly effective expression of heterologous proteins.
