The Construction Of Secretive Fusion Vector Of Goose Parvovirus VP3 Gene And Goose Interleukin-2 Gene And Expression In Lactobacillus

With the development of centralized breed and humanistic management, oral vaccinegradually became one of the research hotspots. Lactobacillus was normal probiotics inintestinal, interleukin-2 was a good endogenous immunostimulants, which had the naturalfunction of antibacteria, antivirus and antiperiodic. Lactobacillus casei has been developedto be a gene engineering bacteria with special roles, and made it release antigen in intestine.This study constructed a fusion protein of goose parvovirus VP3 gene and gooseintedeukin-2 mature protein gene. It would be a good oral Lactobacillus gene engineeringvaccine for the fusion protein of intedeuin-2 and GPV VP3.1. Firstly, we investigated Lactobacilli from intestinal of goose in 20 olddays, the results showed that Lactobacilli had been reached 10~8CFU/g. 15Lactobacilli were isolated and identified, they were L.sanfrancisco, L.fermentus,L.curvatus, L.parakefir, L.L.helveticus, L.bifermentus, L.murinus, Lamylophilus andL.coryniformis sub.sp.coryniformis. The results indicated that L2 and L8 could betolerant to acid and bile-salt, and there was no plasmid in all 15 Lactobacillusisolated.2. A pair of primers were designed by the S-layer protein gene of Lactobacillus brewsregistered in GenBank, then the signal peptide gene of S-layer protein was obtained byPCR(Polymerase Chain Reaction), it was cloned into pMD18-T vector and sequenced. Theresults showed that the sequence was 352bp long, and the latter 90bp which encodingsignal peptide were identical with Z14250 in GenBank. Bioinformatics analysis indicatedthat it was a signal peptide, and it had the typical characteristics of signal peptide. Theresults showed that the signal peptide gene of S-layer protein was obtained.3. The DNA encoding VP3 structure protein of Goose Parvovirus (GPV) CHv strainwas amplified by PCR according to the genome of GPV B strain in GenBank, and clonedinto pUC57 vector and sequenced, the gene and amino acid sequence were compared with other GPV, MDPV, PPV, CPV and BPV, and analyzed by bioinformatics. The resultsshowed that the DNA of VP3 was 1605bp and coded 534 amid acids, it shared 93.4％-99.8％nucleotide sequences identity among GPV VP3, and shared 96.5％～99.3％amino acid sequences identity, which was the reasons why there was only one serum typeof GPV. GPV CHv VP3 and MDPV shared 79.6％nucleotide sequences identity and89.9％amino acid sequences identity, and it shared less 30％nucleotide sequencesidentity with other parvovirus. Bioinformatics analysis showed that it was a hydrophobie,stable and strong antigenic capsid protein.4. Using oligonucleotide primers based on the conserved interleukin-2 sequences ofduck, the nucleic sequences of interleukin-2 gene of Sichuan White Goose (SChGIL-2)was cloned and sequenced by RT-PCR from peripheral blood lymphocyte. The resultsrevealed that SChGIL-2 was 468nt long, which contained an open reading frame(ORF) of441 base pairs encoding a protein of 146aa. The SChGIL-2 amino acids contained 4phosphorylation sites, 1 N-glycosylation site and a signal peptide with 21 amino acidresidues. The nucleic and predicted amino acid sequences of SChGIL-2 gene shared 99.8％,92.2％, 77.5％, 78.2％and 100％, 85.8％, 65.5％, 64.1％identity with goose ZHJ, duck,chicken, turkey, respectively, but was very low homology with other species. Phylogeneticanalysis showed that SChGIL-2 had a close relationship with duck and chicken. The IL2cgene which encoded IL2 mature protein was 407bp long, and included a encoding regionof 126 amid acids. Bioinformatics analysis showed that it was a hydrophilic protein,5. The SP gene and VP3 gene were jointed by restriction enzyme and PCR, and the IL2cgene was jointed with SP-VP3 by a linker peptides of GSGGSGG, then the fusion geneVP3-IL2c was obtained. It was subcloned into the plasmid pMJ67 to construct expressionplasmid pMJ-SP-VP3-IL2c, and it was transformed into L.casei by electrophoresis, thefusion protein was successfully expression with lactose inducer. The molecular weight offusion prtotein was about 76kD by SDS-PAGE analysis, which was consistent to molecularweight deduced. The expression conditions were determined by optimizing time of induceradded, induction time and concentration of lactose. The optimization conditions were 0.5％lactose, 20h induced time, and lactose added when OD600=0.5. The results showed thatthe fusion protein had good immunogenicity with using anti-GPV rabbit sera by western-blotting. The fusion protein was purified by Sepharose 4B chromatography. Thebioactivities of purified fusion protein was detected by MTT method, the results showedthat the index of proliferation lymphoblast with fusion protein was 3.84, which indicatedthat the bioactivities of expressed IL2c protein was obvious.