| Lactic acid bacteria(LAB)are a group of gram-positive,peroxidase-negative bacteria which can generate lactic acid by metabolize carbohydrates in the living environment.Most of them are regarded as food-grade safe bacteria(GRAS).Because of its extensive probiotic effect,relatively mature gene manipulation platform,its own absence of endotoxin and natural adjuvant effect,it is regarded as a promising candidate strain of oral live carrier vaccine.The constitutive expression system can be expressed together with the host bacteria’s own protein expression without any physical and chemical factors.Compared with the induced expression system,it is more suitable for the development of lactic acid bacteria as a live vector vaccine for delivery of foreign proteins.The diseases caused by Aeromonas hydrophila and Cyprinid herpesvirus 2(CyHV-2)have caused serious damage to the aquaculture industry.Especially,there was no effective vaccine to control CyHV-2 infection.This study aims to develop a protective antigen,delivered by lactic acid bacteria as a live carrier,which provide a theoretical basis and technical platform for the development of oral vaccines against aquatic diseases.1 Lactic acid bacteria-Escherichia coli shuttle carrier transformation and analysis of biological characteristics of recombinant lactic acid bacteriaPPG612,as a common low-copy lactic acid bacteria-Escherichia coli shuttle expression vector,needs to add xylose as inducer in the growth process of recombinant lactobacillus to express exogenous proteins,which limits the study of this vector as oral live carrier vaccine.In order to obtain a novel lactic acid bacteria-Escherichia coli shuttle expression vector which can continuously express foreign proteins on the carrier without the addition of inducers,this study reserved the skeleton region of pPG612 vector,replace the original inducible expression cassette with a constitutive expression cassette,and add T7g10 translation in sequence downstream of the PHCE promoter Enhancer,USP45 secretory signal peptide,compact polyclonal cleavage site,T7t,rrnBTland rrnBT2 tandem terminator.The experiment successfully constructed pPG-HCE lactic acid bacteria-Escherichia coli shuttle constitutive expression vector.In addition,the basic biological characteristics of recombinant lactic acid bacteria with the modified vector were analyzed,and the expression level of heterologous protein was evaluated using enhanced green fluorescent protein(EGFP)as a reporter gene.The results show that the pPG-HCE vector can still exist stably without resistance screening conditions.Genetic stability test show that there is no sequence mutation of the vector,and the vector can not affect the growth of the bacteria.Fluorescence quantitative PCR and Western blot show that the target gene was expressed successfully in the host bacteria,and the expression level is significantly higher than that of in pPG612 vector.2 CyHV-2 virus particle purification and its antigenicity analysis of envelope proteinCyHV-2 can cause haemorrhagic disease in crucian carp,which is very harmful to the aquaculture industry.However,there is no stable cell line that can be used to culture the CyHV-2 virus,and there is no commercial vaccine for the prevention and control of the virus infection.In this experiment,PCR identification of CyHV-2 was performed used crucian carp with gill hemorrhage.CyHV-2 virus particles were successfully purified from diseased crucian carp tissues by sucrose density gradient centrifugation,and mouse anti-CyHV-2 virus was prepared,and the antibody titer reached 1:2000.B cell epitope analysis and transmembrane structure prediction of four envelope proteins(ORF32,ORF81,ORF108,ORF131)of CyHV-2 virus revealed that ORF32 and ORF131 have multiple potential B cell epitopes.Four protein prokaryotic expression vectors were constructed using the virion genome as a template.The two envelope proteins ORF32 and ORF131 were successfully cloned and expressed.Western blot confirmed that they could be recognized by CyHV-2 whole virus antibody.Compared with ORF32,ORF131 has a larger molecular weight and a uniform distribution of B cell epitopes,so it is used as a candidate protein for further study.The mice were immunized by ORF131 protein to prepare serum polyclonal antibody,the antibody titer reached 1:4000,which provided a material basis for the detection of ORF131 protein.3 Construction of recombinant Lactobacillus expressing Hcp protein and ORF31 protein and analysis of its immune efficacyThe constructed lactobacillus expression system was used to express the ORF31 protein of cyhv-2 and the hemolysin co-regulatory protein(Hcp)of aeromonas hydrophila,respectively.In addition,ORF131 and Hcp proteins were connected by a flexible junction peptide to achieve the fusion expression of the protective antigen of the two pathogenic microorganisms.Subcellular localization indicated that both Hcp and ORF131 proteins expressed separately could be secreted to the extracellular environment,while the superscript of Hcp-glinker-ORF131 fusion protein was not detected in the culture medium.In order to evaluate the immune effect of recombinant Lactococcus lactis on animal body,in this study,recombinant Lactococcus lactis with Hcp protein,ORF131 protein and Hcp-glinker-ORF 131 fusion protein were expressed by gavage in ICR mice of model animals.SIgA levels and IgG levels in intestinal and fecal tissues and blood were detected by indirect ELISA on 35d and 49d,and transcription levels of blood,spleen and intestinal cytokines were detected by fluorescence quantitative PCR on 35d and 49d to evaluate the immune effect of recombinant Lactobacillus on mice.Indirect ELISA results show that three lactic acid bacteria can well stimulate the body to produce specific sIgA mice and IgG,among them 35 d antibody levels higher than 49 d,lavage Hcp-glinker-ORF131 fusion protein expression of lactic acid milk aureus antibody levels of the experimental group is higher than other groups,each group basic cellular transcription factor test result is consistent with the trend of antibody detection.Studies have shown that recombinant lactobacillus expressing protective antigen can stimulate the body to produce specific antibodies and stimulate the mucosal immune system.In conclusion,two protective antigens were successfully expressed by means of Lactobacillus system,and proved to have good immune effect in animal test.This study laid a foundation for the development of aquatic animal oral vaccines. |
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