人胃内因子在谷氨酸棒杆菌中的表达及活性研究

Expression and optimization of recombinant human gastric factors in Corynebacterium glutamicum

人胃内因子(GIF)在人体内具有重要的作用,主要协助VB12在小肠中的吸收,缺失将导致机体恶性贫血,同时会降低免疫力。谷氨酸棒杆菌(C.glutamicum)作为一种不产内毒素的食品级安全微生物,在药用蛋白的表达上具有巨大的优势。为了在C.glutamicum中进行药用蛋白GIF的表达,编码GIF的重组基因rgif被合成,且进行密码子优化以保证GIF的正常翻译。重组基因rgif成功克隆到表达载体pXMJ19上,并导入C.glutamicum成功进行GIF的表达。为了提高GIF的表达效果,采用正交试验设计对GIF的发酵条件进行优化。发现GIF在C.glutamicum中的最佳发酵条件组合为:IPTG浓度100μmol/L、诱导时机10 h、发酵时长22 h、发酵温度23℃,在该条件下GIF得到可溶性表达,且可以通过His标签被HisTrap FF柱纯化。在谷氨酸棒杆菌中表达的GIF可以通过ELISA试剂盒检测到免疫活性,且定量产量达到42.3 mg/L。根据正交试验的结果分析发现,GIF的产量随着发酵温度从23℃增加到37℃持续降低,随着诱导时机从0 h延迟到10 h持续增加,随着发酵时长从18 h增加到22 h而增加,之后降低,随着诱导剂浓度从50μmol/L增加到100μmol/L而增加,之后降低,且各因素的影响强度依次减弱。因此,根据以上结果可知适当地降低发酵温度、延迟诱导时机将是有效提高GIF可溶性表达的研究方向。研究首次用原核生物表达GIF,为GIF的原核表达提供了一种可行的方案。

The human gastric factor(GIF)is a very important protein,and it plays a very important role in assisting the absorption of VB12 through small intestinal wall cells.If there be synthetic disorder of the protein,it will lead to the occurrence of atypical anemia in the body and reduce immunity.C.glutamicum is a food-grade safe microorganism,no endotoxin is detected in the fermentation broth of C.glutamicum.In addition,it has some unique advantages in its use for protein expression.In order to express pharmaceutical GIF in C.glutamicum,a recombinant gene rgif was synthesized,and codon optimization of the gene sequences to ensure normal translation of rgif into GIF.The recombinant gene rgif was successfully cloned into the expression vector pXMJ19,the vector containing rgif was introduced into C.glutamicum,and the protein GIF was expressed successfully.To improve the yield of GIF,fermentation process was optimized through orthogonal experimental design.The optimal fermentation conditions to express GIF in C.glutamicum were as follows:IPTG concentration was 100μmol/L,time of adding induction was 10 h,fermentation time was 22 h and fermentation temperature was 23℃.Under which,GIF was abundantly expressed in C.glutamicum,and it was soluble.Proteins harvested could be purified by HisTrap FF column.The protein could also be detected by an ELISA kit,and the yield of GIF reached to 42.3 mg/L.According to the results of orthogonal experiments,it was found that the yield of GIF continued to decrease as the fermentation temperature increased from 23℃to 37℃.The yield of GIF was also continuously reduced during the time of adding induction from 0 to 10 h.When it comes to the fermentation time,GIF production increased continuously from 18 to 22 h,but decreased gradually after 22 h.As to IPTG,GIF production gradually increased as its concentration increased from 50 to 100μmol/L,but decreased thereafter.The effect of the above four factors on protein production was weakened in turn.It could be seen that giving a lower temperature,delaying the time of induction would be an effective work to improve the soluble expression of GIF.This was the first report of the expression of GIF by prokaryote,which would provide a viable solution for prokaryotic expression of GIF.