猪流行性腹泻病毒S2基因B细胞表位嵌入型S-层蛋白在副干酪乳杆菌中的表达与鉴定

doi:10.16431/j.cnki.1671-7236.2022.10.033

Abstract

Abstract: 【Objective】 This study was aimed to construct a novel Porcine epidemic diarrhea virus (PEDV) live Lactobacillus vaccine vector.【Method】 The S-layer protein (SLP) gene of Lactobacillus acidophilus and the B cell epitope (EpitopeS2) of PEDV S2 gene fusion gene (SLP-EpitopeS2) were cloned into Lactobacillus sp.expression vector pTRK892 by DNA recombination technique,and the recombinant vector pTRK-SLP-EpitopeS2 was constructed.The recombinant plasmid was introduced into the Lactobacillus paracasei by electric transformation to obtain the recombinant Lactobacillus paracasei.The expression of the target protein in Lactobacillus paracasei was identified by SDS-PAGE,Western blotting and immunofluorescence assay (IFA),respectively.【Result】 A target band of 1 400 bp was successfully amplified by PCR,which was consistent with the size of the inserted fusion gene.Two bands of 1 400 and 4 700 bp were found in the double enzyme digestion assay,which were consistent with the size of the inserted fusion gene and vector fragment.The gene sequencing results showed no base loss or mutation,etc.Thus,the construction of the recombinant plasmid pTRK-SLP-EpitopeS2 was confirmed to be correct.The results of SDS-PAGE and Western blotting showed that the target protein band consistent with the theoretical value appeared at 48 ku,indicating that the fusion gene SLP-EpitopeS2 was effectively expressed in Lactobacillus paracasei.The results of IFA showed that,compared with the control group Lactobacillus paracasei,recombinant Lactobacillus paracasei could be stimulated to give a specific green fluorescent signal,which were consistent with the results of the supplementary identification test for bacterial membrane protein samples eluted by high concentration of lithium chloride (LiCl).These results indicated that the fusion protein SLP-EpitopeS2 might be expressed on the surface of Lactobacillus paracasei.【Conclusion】 The PEDV EpitopeS2 and Lactobacillus acidophilus SLP embedded fusion expression vector pTRK-SLP-EpitopeS2 was successfully constructed,which laid a foundation for the related research on live Lactobacillus carrier vaccine.

Key words: Porcine epidemic diarrhea virus (PEDV); S2 gene; Lactobacillus paracasei; expression; identification

CLC Number:

S852.65^＋1

BAI Weiqin, KA Chula, WU Zhiyong, MIAO Miao, TA Na, GE Riletu. Expression and Identification of B-cell Epitope Embedded S-layer Protein of Porcine Epidemic Diarrhea Virus S2 Gene in Lactobacillus paracasei[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 4004-4011.

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Expression and Identification of B-cell Epitope Embedded S-layer Protein of Porcine Epidemic Diarrhea Virus S2 Gene in Lactobacillus paracasei

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