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

摘要/Abstract

摘要： 【目的】构建新型猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)乳酸菌活菌疫苗载体。【方法】应用DNA重组技术将嗜酸乳杆菌S-层蛋白(SLP)基因及PEDV S2基因B细胞表位(EpitopeS2)融合基因(SLP-EpitopeS2)克隆到乳酸杆菌表达载体pTRK892中,构建重组载体pTRK-SLP-EpitopeS2,通过电转化方法将重组质粒导入副干酪乳杆菌中,获得重组副干酪乳杆菌。分别用SDS-PAGE、Western blotting和免疫荧光试验(IFA)鉴定目的蛋白在副干酪乳杆菌中的表达。【结果】PCR结果显示,成功扩增出大小为1 400 bp的目的条带,与插入融合基因大小一致,双酶切结果出现大小分别为1 400和4 700 bp的2条带,基因测序结果显示无碱基缺失和突变等,从而确定重组质粒pTRK-SLP-EpitopeS2构建正确。SDS-PAGE、Western blotting结果显示,在48 ku处出现与理论值大小一致的目的蛋白条带,表明融合基因SLP-EpitopeS2在副干酪乳杆菌中得到有效表达。IFA结果显示,与对照组副干酪乳杆菌相比,重组副干酪乳杆菌均能被激发出特异性绿色荧光信号,与高浓度氯化锂(LiCl)洗脱下来的菌体膜蛋白样品补充鉴定试验结果相吻合。表明融合蛋白SLP-EpitopeS2可能在副干酪乳杆菌的菌体表面表达。【结论】成功构建了PEDV EpitopeS2及嗜酸乳杆菌SLP嵌入型融合表达载体pTRK-SLP-EpitopeS2,为乳杆菌活菌载体疫苗相关研究奠定了基础。

关键词: 猪流行性腹泻病毒(PEDV); S2基因; 副干酪乳杆菌; 表达; 鉴定

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

中图分类号:

S852.65^＋1

白伟琴, 卡楚拉, 乌志勇, 苗苗, 塔娜, 格日勒图. 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[J]. 中国畜牧兽医, 2022, 49(10): 4004-4011.

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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