关键词: 谷氨酸棒杆菌, 人胃内因子, 表达, 正交优化, ELISA

Abstract: 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 100 μmol/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 100 μmol/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.

Key words: Corynebacterium glutamicum, human gastric factor, expression, orthogonal optimization, ELISA