表达A亚群禽白血病病毒Gp85蛋白的乳酸菌的构建

doi:10.16431/j.cnki.1671-7236.2021.09.038

Abstract

Abstract: The aim of this study was to construct a lactic acid bacteria (LAB) expression vector to develop a live bacterial vaccine of Avian leukosis virus subgroup A (ALV-A) for the prevention of avian leukemia. In this experiment, an anchored expression vector was used to construct two recombinant Lactobacillus plantarum (L. plantarum) expressing gp85 and gp85-DCpep genes. Using the genomic DNA of DF-1 cells infected with ALV-A as a template, PCR was used to amplify the encoding gene gp85 of ALV-A Gp85 protein, then the DC-targeting peptide (DCpep) encoding gene and gp85 gene were fused by the same method to obtain the gp85-DCpep gene. Then the recombinant plasmids pSIP409-pgsA’-gp85-DCpep and pSIP409-pgsA’-gp85 were constructed by seamless cloning. After sequencing identification, the plasmids were electroporated into L. plantarum NC8 competent cells to obtain recombinant L. plantarum. After induction by SppIP, the cells in logarithmic growth phase were collected. The protein samples of cell membrane were obtained by reverse freezing and thawing. Afterwards, the expression products were analyzed and identified by Western blotting. The results showed that the gene fragments gp85-DCpep and gp85 were successfully obtained while there was no mutation or loss in the recombinant plasmids pSIP409-pgsA’-gp85-DCpep and pSIP409-pgsA’-gp85. All of them were successfully transferred into L. plantarum NC8 competent cells. The expression products were detected by SDS-PAGE and Western blotting, and the bands were visible at 48.3 ku for pgsA’-gp85-Dcpep and at 47 ku for pgsA’-gp85. In this study, the recombinant plasmids pSIP409-pgsA’-gp85-DCpep and pSIP409-pgsA’-gp85 were successfully constructed. The recombinant L. plantarum NC8-pSIP409-pgsA’-gp85-DCpep and NC8-pSIP409-pgsA’-gp85 were obtained and successfully expressed as well as had the reactionogenicity, which laid a good foundation for further study on the protective mechanism of the two recombinant L. plantarum strains against ALV-A infection.

Key words: Avian leukosis virus subgroup A (ALV-A); Gp85 protein; Lactobacillus plantarum; dendritic cells; anchored expression

CLC Number:

S852.65

LI Dingwei, GAO Keli, ZENG Yang, FANG Chun, YANG Yuying, LIU Jing, LIANG Xiongyan. Construction of Lactic Acid Bacteria Expressing Avian Leukosis Virus Subgroup A Gp85 Protein[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3456-3463.

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Construction of Lactic Acid Bacteria Expressing Avian Leukosis Virus Subgroup A Gp85 Protein

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