DNA shuffling approach for recombinant polyvalent OmpAs against V. alginolyticus and E. tarda infections.
DNA shuffling approach for recombinant polyvalent OmpAs against V. alginolyticus and E. tarda infections.
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DOI：
10.1016/j.fsi.2016.09.058
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发表时间：
2016-11
影响因子：
4.7
通讯作者：
Hui Li;Xiao Chu;Bo Peng;Xuan-xian Peng
中科院分区：
文献类型：
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作者：
Hui Li;Xiao Chu;Bo Peng;Xuan-xian Peng
通过DNA改组的分子育种指导具有所需性状的疫苗的进化。本研究首先利用VA 0764引物构建了一个新的OmpA杂合基因，并构建了含有84个OmpA的原核表达文库PompAs-FV，经PCR和SDS/PAGE验证，获得了含有84个OmpA的多价OmpA疫苗。然后，利用这84个ompAs构建了用于制备DNA疫苗的真核表达文库EompAs-FV。第三，用这些DNA疫苗进行主动免疫后的胞外细菌攻击，以鉴定具有高免疫保护性的基因。在84个ompA中，17个显示出比对照VA 0764更高或相等的针对由溶藻弧菌引起的感染的免疫保护。最后，利用17个ompAs中的前7个ompAs进一步评价了针对细胞内细菌迟缓爱德华氏菌感染的免疫保护作用，从而鉴定出三种有效的针对细胞外细菌溶藻弧菌和细胞内细菌迟缓爱德华氏菌感染的多价疫苗。但免疫保护与基因突变和氨基酸突变的关系尚不明确。这些结果表明，DNA改组是一种有效的方法来开发针对微生物感染的多价疫苗。
Molecular breeding via DNA shuffling directs the evolution of vaccines with desired traits. In the present study, polyvalent OmpA vaccines were generated by DNA shuffling of fiveompAgenes from four species of bacteriaVibrio parahaemolyticus, V. alginolyticus, Edwardsiella tardaandEscherichia coli.First, a new hybrid OmpA was constructed using VA0764 primers and used for construction of a prokaryotic expressing library PompAs-FV containing 84ompAs, which were validated by PCR and SDS/PAGE. Then, the 84ompAswere used to construct a eukaryotic expressing library EompAs-FV for preparing DNA vaccines. Third, extracellular bacteriumV. alginolyticuschallenge post active immunization using these DNA vaccines was carried out to identify genes with high immunoprotection. Among the 84ompAs, 17 showed higher or equal immune protection against infection caused byV. alginolyticusthan control VA0764. Finally, immune protection against infection caused by intracellular bacteriumEdwardsiella tardawas assessed further using the top seven out of the 17ompAs.This led to identification of three efficient polyvalent vaccines against infections caused by the extracellular bacteriumV. alginolyticusand intracellular bacteriumE. tarda.In addition, we sequenced genes for understanding mechanisms of the polyvalent vaccines, but association of immune protection with mutation of gene and amino acids is not determined. These results indicate that DNA shuffling is an efficient way to develop polyvalent vaccines against microbial infections.