The Tandem Expressing Of The Antigen Epitope Genes Of RHDV-VP60 And Pm OmpH?OmpA And Study On Their Immunogenicity In Rabbit

Rabbit hemorrhagic disease and Pasteurellos is belong to important infectious diseases which have a great threat on the development of rabbit industry,respectively caused by Rabbit hemorrhagic disease virus?RHDV?and Pasteurela multocida?Pm?.These diseases have high incidence and mortality.In clinical,diseases showed an phenomenon of acute onset and mixed infection,huge economic losses happend on many farms while occurring.At present,the vaccines aimed to prevent and control diseases were mainly focused on bicombinant inactivated vaccine of Rabbit hemorrhagic disease and Pasteurellosis.The kind of vaccine has disadvantages of massive amounts and production of humoral immunity,so it is necessary to develop a new-type vaccine.Multiple epitope vaccine is a new kind of subunit vaccine,which has been studied by biotechnology in recent years.It carried multiple target antigens and had characteristics of multiple prevention to one vaccine and good security.The aim of this study is to construct the prokaryotic expression vector containing dominant epitope region of the main protective antigen for RHDV and Pm,then study their immunogenicity within body of rabbit,providing a new idea for development of a multiple epitope vaccine which facilitate for prevention and control of Rabbit hemorrhagic disease and Pasteurella.Based on isolation and identification of RHDV and Pm strain,sequences of RHDV VP60 gene and OmpH and OmpA gene of Pm were amplified by PCR respectively.Antigen epitopes of these three proteins were analyzed by BepiPred-2.0 online tool,regions combining strong antigenicity were screened and connected in series by flexible connecting peptide Linker?Gly₄Ser?₃.The gene encoding this tandem epitope is optimized according to codon preference of Escherichia coli to synthesize.The prokaryotic vector of pET30a?+?-3epi was constructed and transferred into the Escherichia coli BL21 for prokaryotic expression;the recombinant protein was purified and identified.The purified recombinant protein was used to immunize rabbits to study the effect of immune protection.The results as follows:1.Identification of RHDV SZ/2016 and its VP60 sequence analysisIn 2016,samples were collected from a suspect RHD rabbits in rabbits warren of Shizhu Chongqing,VP60 gene in the sample were detected by RT-PCR and about 1.7kb fragments were obtained.The purified PCR products were sent to the company for sequencing.The results showed that total length of VP60 gene encoding 579 amino acids is 1740 bp.Compared with the VP60 nucleotide sequence of 16 RHDV strains published on GenBank,homology reached 94.9⁹5.9%,which proved to be Rabbit hemorrhagic disease virus?SZ/2016?.Hemagglutination test indicated that HA titer of RHDV?SZ/2016?strain was 1:320.A 2kg of healthy rabbit was attacked by 1 mL RHDV?SZ/2016?strain?1:10 liver tissue suspension?,the rabbits died from 20 h to 48h,manifesting strong virulence of RHDV?SZ/2016?strain.The VP60 DNA sequence was translated into amino acid sequence,and BepiPred-2.0 online tool was used to predict the epitopes of VP60 protein.The results displayed that B cell epitope of VP60 protein was mainly located at the 4⁸6 and230²58 amino acid regions.2.Isolation of Pasteurella multocida and analysis of OmpH and OmpA gene sequencesIn 2016,samples were collected from a suspect Rabbit pasteurellosis rabbits in warren of Shizhu Chongqing,The isolateed strains were identified as Pasteurella multocida?Pm SZ/2016?via identification of bacteria isolation,morphology,biochemistry and 16S rRNA.The phylogenetic tree of 16S rRNA gene of Pm?SZ/2016?was constructed,which indicated that Pm?SZ/2016?strain and 12 reference strains published on GenBank could be divided into two families,clustered with KX579747.1and MG597100.1.The pathogenicity test of rabbit showed that the minimum lethal dose?MLD?of Pm?SZ/2016?strain in rabbits was 10 CFU/mL×1mL.The OmpH and OmpA gene of Pm?SZ/2016?were amplified by PCR.The purified PCR products were sent to sequence by company.Results showed that OmpH gene sequence was 1 056 bp and 351 amino acids were encoded.Compared with the OmpH nucleotide sequence of 16 Pm strains published on GenBank,homology reached 98 to99%.The OmpA gene sequence was 1 062 bp,encoding 353 amino acids.Compared with the OmpA nucleotide sequence of 14 Pm strains on GenBank,the homology reached 96%to 100%.The sequencing OmpH and OmpA DNA sequence was translated into amino acid sequence respectively,BepiPred-2.0 online tool was used to predict epitopes of OmpH and OmpA protein.The results showed that B cell epitopes of OmpH protein were mainly located in 25⁵6,121²16,223²80,326³40 of amino acid regions,B cell epitopes of OmpA protein were mainly located in the 38⁶3,83¹22,139²31,315³40 of amino acid regions.3.Construction of expression vector pET30a?+?-3epi and prokaryotic expression of recombinant proteinBased on the analysis of the epitopes of VP60 of RHDV?SZ/2016?and OmpH/OmpA of Pm?SZ/2016?,epitope regions containing strong antigenicity of three proteins were screened.The 1¹02 amino acid region of VP60 protein,121²16 amino acid region of OmpH protein and 139²31 amino acid region of OmpA protein were selected respectively.Flexible Linker was used to connect dominant epitope regions according to OmpA?aa139 to 231?-Linker-VP60?aa1 to 102?-Linker-OmpH?aa121 to216?.The synthesized sequence has a total length of 978bp,which encodes 323 amino acids.The synthesized DNA sequence was connected on the expression vector pET30a?+?and transformed into Escherichia coli BL21.The successful construction of pET30a?+?-3epi was by identification of PCR,restriction enzyme identification and sequencing.The positive bacteria were induced to express,SDS-PAGE results showed that about 46 Ku recombinant protein of a relative molecular mass had expressed in Escherichia coli successfully.The purified protein was analyzed by SDS-PAGE test and results showed that there was only a clear strip on the gel.Western-blot detection showed that recombinant protein could be identified by RHDV and Pm positive serum.4.Study on immunogenicityThe 20 day old healthy rabbits were immunized the recombinant protein mixed with Freund's adjuvant,and each rabbit was treated with recombinant protein of 0.25mg.Rabbit blood was collected at 14,28 and 42 days after immunization and serum was separated respectively.The antibody levels of RHDV and Pm in serum separated at different times were detected by RHDV and Pm ELISA kit respectively.The results showed that the rabbits in the experimental group produced anti RHDV and Pm specific antibodies two weeks after the first immunization.The antibody level increased with different time and reached to the peak on 28 day and remained stable.The results of attack test on rabbits on the fourteenth day after the third immunization showed that rabbits in the experimental group were able to resist 1 mL RHDV?SZ/2016?suspension with a hemagglutination titer of 1:320 and minimum lethal dose of Pm?SZ/2016?,protective rate could reach 81%and 63%respectively.In summary,based on a rabbit hemorrhagic disease virus and a Pasteurella multocida were obtained,sequences of VP60 of RHDV?SZ/2016?and OmpH/OmpA of Pm?SZ/2016?we analyzed and multi-epitope protein containing the major protective antigens of RHDV and Pm expressed successfully.Animal immunization test showed that recombinant protein could induce rabbit to produce neutralizing antibodies against these two pathogens simultaneously.This study provides a new idea to develop multi epitope vaccines of RHDV and Pm.