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

摘要/Abstract

摘要： 本研究旨在构建乳酸菌表达载体以研制A亚群禽白血病病毒（Avian leukosis virus subgroup A，ALV-A）的活菌疫苗，用于预防禽白血病。使用锚定表达载体构建2株分别表达gp85和gp85-DCpep基因的重组植物乳杆菌，以感染ALV-A的DF-1细胞的基因组DNA为模板，使用PCR方法扩增ALV-A Gp85蛋白的编码基因gp85，将树突状细胞靶向肽（DC-targeting peptide，DCpep）编码基因与gp85基因进行融合，获得gp85-DCpep基因；使用乳酸菌锚定表达载体pSIP409-pgsA’通过同源重组的方法构建重组质粒pSIP409-pgsA’-gp85-DCpep和pSIP409-pgsA’-gp85；经测序正确后将上述质粒分别电转化植物乳杆菌NC8感受态细胞，获得重组植物乳杆菌；使用SppIP诱导重组植物乳杆菌的表达，收集处于对数生长期的菌体，通过反复冻融获取细胞膜的蛋白样，采用Western blotting技术检测pgsA’-gp85-DCpep和pgsA’-gp85蛋白的表达情况。结果显示，试验成功获得基因片段gp85和gp85-DCpep，构建的重组质粒pSIP409-pgsA’-gp85-DCpep和pSIP409-pgsA’-gp85测序无突变和丢失情况，并成功电转到植物乳杆菌NC8感受态细胞中，使用SDS-PAGE和Western blotting技术在细胞膜蛋白样中检测到蛋白的表达，大小分别为48.3（pgsA’-gp85-DCpep）和47 ku （pgsA’-gp85）。本试验成功构建了重组质粒pSIP409-pgsA’-gp85-DCpep和pSIP409-pgsA’-gp85，获得的重组植物乳杆菌NC8-pSIP409-pgsA’-gp85-DCpep和NC8-pSIP409-pgsA’-gp85中的蛋白均成功表达，且具有反应原性，为后期深入研究重组植物乳杆菌在抗ALV-A感染中的保护机制奠定良好的基础。

关键词: A亚群禽白血病病毒(ALV-A); Gp85蛋白; 植物乳杆菌; 树突状细胞; 锚定表达

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

中图分类号:

S852.65

李鼎伟, 高可丽, 曾扬, 方春, 杨玉莹, 刘晶, 梁雄燕. 表达A亚群禽白血病病毒Gp85蛋白的乳酸菌的构建[J]. 中国畜牧兽医, 2021, 48(9): 3456-3463.

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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