Preliminary Application Of Recombinant Lactobacillus Of Grass Carp Hemorrhagic Disease Virus VP7 Gene

Grass carp hemorrhage is an important disease caused by grass carp reovirus?GCRV?,which has strong transmission ability and high mortality rate,which seriously endangers the development of grass carp industry.At present,there are almost no drugs for treating grass carp bleeding,and long-term use of drugs is likely to cause problems such as water pollution and drug resistance.Vaccine immunization is a feasible solution to prevent grass carp hemorrhage,but because the injection immunization operation is cumbersome,it is easy to cause damage to the fish body and produce stress,soaking immune effect is poor and it is easy to cause environmental pollution.There is an urgent need to develop a solution that is suitable for operation and has an excellent immune effect.Lactic acid bacteria?LAB?is a recognized food-grade safe microorganism.The development of oral vaccines as a carrier is a new field and new hotspot for the application of lactic acid bacteria.In this study,cloning grass carp hemorrhagic disease virus coat protein VP7 was ligated to the vector and transformed into Lactobacillus plantarum NC8 to prepare lactic acid bacteria-mediated oral vaccine for grass carp hemorrhage,and the immune effect was evaluated by oral feeding.Five subgroups were set up in the experiment,1.0×10⁹ CFU/g feed concentration group,1.0×10⁸ CFU/g feed concentration group,1.0×10⁹ CFU/g empty carrier group,1.0×10⁸ CFU/g empty carrier group and blank control group,and the feed amount was fed.The feeding was carried out according to3%of the body weight,and the feeding was performed once a day for 3 days.The immunization was boosted in the same way two weeks later.Serum,liver,and kidney were collected on the 1 d,3 d,7 d,12 d,16 d,21 d,31 d and 38 d after the end of immunization.The level of specific antibody IgM in serum was determined by ELISA,the gene expression of immune related genes IFN1,IL-1?and MHC-IIB was determined by RT-PCR,the activity of lysozyme and POD and SOD in the liver was determined by the test kit.Intestinal tissue was taken to study the colonization of recombinant lactic acid bacteria in the intestinal tract of grass carp.The results obtained are as follows:The ELISA results show:The level of IgM in the serum of the oral immunization?pSIP409-vp7?group increased first and then stabilized,and was significantly higher than that of the empty vector?pSIP409?group and the blank control?PBS?group?P<0.05?.It peaked at 12d(OD₄₉₀:1.29;OD₄₉₀:1.19).At the same time,the antibody level of the 1.0 x 10⁹ CFU/g oral immunization?pSIP409-vp7?group was higher than the 1.0 x 10⁸ CFU/g oral immunization?pSIP409-vp7?group.RT-PCR results show:After oral lactic acid bacteria vaccine,the expression levels of IL-1?,IFN I and MHC-II B in the kidneys of the two concentrations were increased.The expression level of oral immunization?pSIP409-vp7?group was significantly higher than that of empty vector?pSIP409?group and blank control?PBS?group.The expression level of1.0×10⁹ CFU/g oral immunization?pSIP409-vp7?group was significantly higher than that of other groups?P<0.05?.The results of non-specific immune indicators showed:Oral lactic acid bacteria recombinant vaccine can significantly increase the activity of superoxide dismutase?SOD?,peroxidase?POD?and lysozyme?LZM?in the blood and liver of grass carp.The activity was significantly higher than that of the blank control?PBS?group?P<0.05?.The SOD,POD and LZM activities of the oral immunization?pSIP409-vp7?group in both concentration groups peaked at 7-12 d.The results of intestinal colonization test showed that the recombinant lactic acid bacteria were mainly colonized in the intestine of the grass carp,and the colonization time was 7-12 d The above results indicate that the recombinant lactic acid bacteria vaccine can significantly increase the antibody level of grass carp IgM and the level of non-specific immune indicators.It can also promote the expression level of immune-related genes,thereby improving the immune response level of grass carp.The results can provide new ideas for the prevention and control of other aquatic animal related viruses.