共表达鹅细小病毒VP3-gIL2重组乳酸杆菌的构建及其口服免疫评价

Construction of Recombinant Lactobacillus Co-expressed GPV VP3-gIL2 and Oral Immune Effect to Goose

笔者拟构建共表达鹅细小病毒(GPV)VP3和鹅白细胞介素-2(IL-2)的分泌性重组乳酸杆菌,评价口服表达融合蛋白GPV VP3-gIL2的重组乳酸杆菌的免疫效果。通过酶切将VP3基因和IL-2基因连接,并在其5′端连入乳酸杆菌的信号肽基因SP,亚克隆到乳酸杆菌整合表达载体pMJ67,电转化入干酪乳杆菌L.CECT5276,SDS-PAGE和Western blot法鉴定重组菌GPVVP3-gIL2融合蛋白的表达活性。将重组乳酸杆菌口服免疫雏鹅后检测血清GPV抗体、小肠黏液sIgA和脾淋巴细胞增殖情况,评价其免疫效果。结果表明,酶切和测序结果表明成功构建重组表达载体pMJ-SP-GPVVP3-gIL2,PCR证实质粒pMJ-SP-GPVVP3-gIL2成功整合到乳酸杆菌基因组;Western blot表明重组乳酸杆菌分泌表达的融合蛋白GPVVP3-gIL2能与GPV阳性血清特异性结合;淋巴细胞增殖试验表明口服重组菌后能特异地刺激脾淋巴细胞增殖,且在第4、5、6周明显高于GPV VP3组和gIL2组;重组乳酸杆菌口服免疫雏鹅可以产生抗VP3蛋白抗体,且具有中和活性的抗体显著高于GPV VP3组;小肠黏液sIgA细胞生成的数量自第2周较GPV VP3组和疫苗组显著上升。动物攻毒试验结果显示GPV VP3-gIL2融合蛋白免疫组能够获得85%的保护率,表明所构建的重组乳酸杆菌口服后对GPV的攻击具有较好的免疫保护作用。本试验成功构建能稳定表达GPV VP3-IL2融合蛋白的口服乳酸杆菌工程菌,为研究其作为口服疫苗奠定了基础。

The aim of this study was to construct a recombinant secreted Lactobacillus oral live vector strain co-expressing goose parvovirus（GPV） VP3 protein and goose interleukin-2（gIL-2） protein.The VP3 gene was ligated with gIL-2 by enzyme digestion,and the signal peptide（SP） gene from Lactobacillus brevis 1.2028 was located on the N-terminal of fusion protein.The three genes（as a complete open reading frame） were subcloned into Lactobacillus integrated expression vector pMJ67,and then the recombinant plasmid pMJ-SP-GPVVP3-gIL2 was electro-transformed into Lactobacillus casei CECT5276 to construct the oral live vector vaccine strain.The recombinant Lactobacillus was identified by PCR method and immunogenicity of expression protein was tested by SDS-PAGE and Western blot methods.The specific anti-GPV antibody in serum,secretive immunoglobin A（sIgA） and spleen lymphocyte proliferation were detected to evaluate the immune effects after oral immunization goose with the recombinant Lactobacillus.The results showed that the recombinant plasmid has been constructed successfully,PCR proved that the plasmid pMJ-GPVVP3-gIL2 has been integrated into Lactobacillus genome,SDS-PAGE and Western blot could detect the GPV VP3-gIL2 protein band in supernatant and it could be recognized by the positive serum against GPV.Spleen lymphocyte proliferation responses results indicated that recombinant Lactobacillus could also induce an obvious cellular immune response,the lymphocytes proliferation level of GPV VP3-gIL2 group was obviously higher than GPV VP3 group and gIL2 group in the 4th,5th and 6th week.Neutralization antibodies could be produced in GPV VP3-gIL2 group,GPV VP3 group and gIL2 group,but the neutralization antibodies level of GPV VP3-gIL2 group was significantly higher than that of the latter two groups.Since the 2nd week,the number of sIgA in intestinal mucosal fluid was significantly higher than GPV VP3 group and Vaccine group.Challenge experiment results showed that goose immunized with the GPV VP3-gIL2 fusion protein obtained 85% protection;the results confirmed that the recombinant Lactobacillus could enhance the protection against GPV.A recombinant oral live vaccine Lactobacillus CECT5276（pMJ-GPVVP3-gIL2） secretive expressing fusion protein GPV VP3-gIL2 was constructed successfully,this recombinant Lactobacillus could be a basis to oral live vector vaccine for GPV infection.