Tβ4②蛋白基因工程菌发酵条件的优化研究

Optimization of the Conditions of Fermentation by Orthogonal Test for the Engineering Bacterial Strain with Thymosin Beta Dimer Protein Gene

通过正交实验对重组胸腺素β4②基因工程菌的发酵条件进行优化。选择温度、接种量、诱导剂浓度和诱导时机4因素,分别在3个水平上进行考察。结果显示影响Tβ4②菌体浓度的主次因素顺序:诱导时机>温度>诱导剂浓度>接种量,影响Tβ4②表达水平的主次因素顺序:诱导时机>接种量>温度>诱导剂浓度,最终确定最佳工艺参数为:诱导时机3 h,温度37℃,IPTG 0. 5 mmol/L,接种量5%,并在5 L发酵罐中进行验证,最终菌体得率为49 g/L,Tβ4②表达量达30%。

Thymosin beta 4( Tβ4),acting as a major regulatory molecule in mammalian cells,plays an important role in the regeneration,remodeling,and healing of injured or damaged tissues. However,it cannot be expressed in E. coli directly because of its low molecular weight. Based on the optimization of the Tβ4 structure,the author constructed recombinant genetically engineered bacteria p ET22 b( +)-Tβ4②/BL21( DE3) and induced with IPTG. The results showed that Tβ4② is able to promote lymphocyte differentiation and proliferation as effectively as synthetic one,and has weak immunogenicity detected by ELISA. However,in the process of expressing Tβ4②,the p ET22 b( +)-Tβ4 ②/BL21( DE3) are affected by many factors,and the culture and expression conditions need to be optimized in many aspects to achieve to the higher yield of bacteria and the efficient expression of recombinant proteins.To optimize the fermentation conditions of p ET22 b-Tβ4②/BL21( DE3),the final bacterial yield of p ET22 b-Tβ4②/BL21( DE3)and the expression level of Tβ4 ② were identified as the evaluation index. The influence of different culture conditions based on shake flask fermentation such as induction time,inoculation proportion,culture temperature and IPTG concentration were investigated by using single factor experiment and orthogonal experiment. The order of primary and secondary factors affecting the density of bacteria: induction time > induction temperature > IPTG concentration > inoculation proportion,the order of primary and secondary factors affecting the expression level: induction time > inoculation proportion > culture temperature > IPTG concentration. The optimal ferment conditions were induction time of 3 h,induction temperature of 37 ° C,IPTG concentration of 0. 5 mmol/L,and inoculation proportion of 5%. Under these conditions,the final bacterial yield of p ET22 b-Tβ4②/BL21( DE3) and the expression level of Tβ4②all reached to the highest level,with the yield and protein expression of 49 g/L and 30%,respectively. This study was meaningful for the production and application of Tβ4② for further research.