摘要
基于毕赤酵母基因组偏好密码子数据表对编码Aii A蛋白的碱基序列进行密码子优化,并构建多拷贝重组Oaii A表达载体p PICZαB-x Oaii A(x=1,2,3),成功表达出具有活性的Aii A蛋白.实验结果表明,优化后Oaii A基因的表达量(2.64 mg·L-1)较优化前aii A的表达量(1.38 mg·L-1)提高约两倍.优化后二拷贝和三拷贝表达量分别为2.84 mg·L-1和2.93 mg·L-1.结果表明密码子优化可以有效提高Aii A蛋白在毕赤酵母中的表达,但是多拷贝表达载体对Aii A的表达促进作用并不显著.提示未来在探索Aii A蛋白在毕赤酵母中的高表达时,通过增加拷贝数来提高产量并不是一个很好的途径,而进行密码子的优化可以有效提高产量.
Many animal and plant diseases are closely related with N-acyl-homoserine lactone（ AHL） which produced by gram-negative bacteria. Aii A protein was encoded by Bacillus thuringiensis which can hydrolyze lactone bond of AHL,thus performs a positive role on reducing animal and plant diseases. Auto inducer inactivation A（ aii A） gene encodes a protein named as Aii A which is an AHL-lactonase,and in order to improve the expression of Aii A protein in Pichia pastoris,this study optimized the amino acid codon of aii A gene according to the data of the genome preference codon of Pichia pastoris. Based on the optimized gene Oaii A,the multicopy recombinant expression vector of p PICZαB-x Oaii A（ x = 1,2,3） was constructed and the functional Aii A was expressed. The results showed that the expression level of Aii A protein from the optimized gene Oaii A（ 2. 64 mg·L- 1） increased almost twofold in comparison with that from the native gene aii A（ 1. 38mg·L- 1）. The expression levels of two and three copies Oaii A gene in Pichia pastoris was 2. 84mg·L- 1and 2. 93 mg·L- 1,respectively. Our results suggest that codon optimization of aii A gene can improve the expression of Aii A protein in Pichia pastoris significantly,but enhance the copy of interested gene in expression vector is not an effective way to improve the yield of Aii A.
出处
《福建师范大学学报（自然科学版）》
CAS
CSCD
北大核心
2015年第6期55-63,共9页
Journal of Fujian Normal University：Natural Science Edition
基金
福建省自然科学基金资助项目(2012J01123)
福建省教育厅资助项目(JK2012009)
