鲤Rhbdd3重组乳酸菌微生物制剂的开发及效果评价

DEVELOPMENT AND IMPACT ASSESSMENT OF RECOMBINANT LACTOCOCCUS LACTIS MICROBIAL AGENT FOR COMMON CARP(CYPRINUS CARPIO)Rhbdd3 PROTEIN

鱼类Rhbdd3(Rhomboid domain-containing protein 3)蛋白具有抗水生病毒感染功能,然而生产实践中缺乏其大量制备及应用方案。为研制鲤Rhbdd3蛋白重组微生物制剂,利用乳酸菌NICE表达系统,构建了Rhbdd3蛋白重组表达质粒pNZ8148-rhbdd3,并转化获得了重组乳酸乳球菌pNZ8148-rhbdd3 NZ9000。利用乳酸链球菌素nisin进行Rhbdd3蛋白的诱导表达,并通过SDS-PAGE和Western blotting进行验证。进一步,将重组乳酸乳球菌进行饲料拌喂并开展鲤春病毒血症病毒(SVCV)人工感染实验,检测鱼体免疫因子表达水平及攻毒后鱼体组织病毒载量。结果显示:经nisin诱导后,pNZ8148-rhbdd3 NZ9000能够表达约为39 kDa的特异性蛋白,与Rhbdd3蛋白大小一致。重组乳酸乳球菌的最佳nisin诱导浓度为100 ng/mL,最佳诱导时间为5 h。重组乳酸乳球菌饲喂后14 d,与对照组相比,实验组鲤鱼组织中免疫相关基因IFN1、IRF7、Viperin、Mx1和ISG15的表达水平均显著上调。此外,SVCV攻毒后,重组乳酸乳球菌饲喂组鱼体组织中病毒滴度较对照组显著降低,表明其可以抑制病毒的增殖。研究结果为Rhbdd3蛋白的大量制备和应用提供了一种可行方案,并为鱼类病毒性疾病的防控提供了一种有效措施。

Studies have shown that fish rhomboid domain-containing protein 3(Rhbdd3)can inhibit aquatic virus infection.However,there is still lack of related application in practice.The Lactococcus lactis NICE(nisin controlled gene expression system)is a high-efficient,safe,and economic protein expression system.For the development of recombinant Lactococcus lactis expressing Rhbdd3,the rhbdd3 gene of common carp was cloned into pNZ8148 vector.The correctness of recombinant plasmid pNZ8148-rhbdd3 was verified by sequencing before electrotransformed into L.lactis NZ9000 competent cells.The recombinant Rhbdd3 protein was induced in different concentrations of nisin for 1,3,5 h and then analyzed by SDS-PAGE and the Western blotting.Then,the verified recombinant L.lactis were added to the base feed and fed to the common carp daily.The fish were then infected with spring viremia of carp virus(SVCV)two weeks later.The relative expression of immune-related genes and the viral N gene copies were detected by RT-qPCR.Results show that the molecular weight of expressed protein was 39 kDa,which is equal to the expected protein size.The optimal condition of induction was 100 ng/mL nisin for 5 h.The expression of immune-related genes including IFN1,IRF7,Viperin,Mx1,and ISG15 Viperin were all up-regulated compared with the control group at 14 days post feeding.In addition,the replication of SVCV in experimental group was significantly lower than that of the control group,indicating that the recombinant L.lactis enhanced the immunity of the common carp.The present study provided a strategy for the large-scale preparation of Rhbdd3 protein as an effective prevention and control measure against aquatic viral disease.