Construction And Application As A Vehicle For PRRSV ORF5-ORF6 Of ?crp?cya?asd C78-1 Balanced Lethal System

Salmonella choleraesuis is an important zoonotic pathogen which causes piglet’s paratyphoid and food poisoning in humans. Mutant strains of Salmonella obtained by deletion attenuation of genetic engineering not only can serve as efficient attenuated vaccine,but also can be developed into a live vehicle for presenting a variety of exogenous protective antigens after introduction of the asd nonresistant balanced lethal system。Screening of suitable attenuated strains to construct efficient vector has been one of the hotspots of vaccine study.Firstly the cya gene was deleted on the basis of the Salmonella choleraesuis Δcrp C78-1 single deletion strain by the method of suicide plasmid-mediated two-step homologous recombination to construct a double deletion strain ΔcrpΔcya C78-1 with weaker virulence and good immunogenicity. Next the asd gene was deleted to develop a balanced lethal system. Finally the system was used as a vector to construct the recombinant attenuated Salmonella choleraesuis strain co-expressing PRRSV protective antigen genes ORF5 and ORF6, which laid a good foundation for development of a bivalent vaccine against piglet paratyphoid and porcine reproductive and respiratory syndrome at the same time. The main contents include:1. Construction and identification of attenuated Salmonella choleraesuis double deletion strain ΔcrpΔcya C78-1The upper and lower arms cya1 and cya2 of cya gene were amplified using Salmonella choleraesuis Δcrp C78-1 single deletion strain as template, then both of them were cloned into the intermediate transfer vector p Bluescript SK(+). Next the tandem fragment cya1 + cya2 was inserted into suicide plasmid p RE112. Finally the recombinant suicide plasmid p REΔcya was introducted into the host E.coli χ7213. E.coli χ7213(p REΔcya) and Δcrp C78-1 were used as donor and receptor respectively, and the Salmonella choleraesuis double deletion strain ΔcrpΔcya C78-1 was screened by the method of suicide plasmid-mediated two-step homologous recombination.2. The biological characteristics of attenuated Salmonella choleraesuis double deletion strain ΔcrpΔcya C78-1Biological characteristics of Salmonella choleraesuis double deletion strain ΔcrpΔcya C78-1 including biochemical property, growth rate, stability of Δcya, toxicity in mice, immunoprotection were studied preliminarily and compared withthose of the single deletion strain Δcrp C78-1, Δcya C78-1 and vaccine strain C500. The results showed that ΔcrpΔcya C78-1, Δcya C78-1 and Δcrp C78-1 had the same ability to utilize carbon sources. They retained the ability to utilize glucose, can’t take full advantage of galactose, and completely lost the ability to use maltose, rhamnose, xylose and other carbon sources; The mean generation time of ΔcrpΔcya C78-1 was 48.2min. The oral LD50 of ΔcrpΔcya C78-1 was 204 times higher than that of Δcrp C78-1,322 times higher than that of vaccine C500; Mice immuned with 109 CFU of ΔcrpΔcya C78-1 were fully protected against challenge with 10 times the LD50 of the wild-type strain.3. Construction and identification of attenuated Salmonella choleraesuis balanced lethal system ΔcrpΔcyaΔasd C78-1E.coli χ7213 carrying recombinant suicide plasmid p REΔasd and attenuated Salmonella choleraesuis double deletion strain ΔcrpΔcya C78-1 were used as donor and receptor respectively, and the Salmonella choleraesuis three deletion strain ΔcrpΔcyaΔasd C78-1 was screened by the method of suicide plasmid-mediated twostep homologous recombination. PCR and sequencing results showed ΔcrpΔcyaΔasd C78-1 three deletion mutant was successfully constructed. In addition, the plasmid p YA3493 containing asd gene was successfully electroporated into the three deletion strain, then the balanced lethal system ΔcrpΔcyaΔasd C78-1(p YA3493) was constructed. The resulting strain were able to grow normally on an ordinary medium without DAP.4. Construction and identification of recombinant attenuated Salmonella choleraesuis ΔcrpΔcyaΔasd C78-1(p YA-ORF5-ORF6)PRRSV ORF5 and ORF6 genes were amplified by RT-PCR, and both of them were inserted into prokaryotic expression vector p YA3493. After identification by PCR, double digestion and sequencing, the recombinant vector p YA-ORF5-ORF6 was introducted into attenuated Salmonella choleraesuis three deletion strain ΔcrpΔcyaΔasd C78-1. PCR and double digestion results showed that recombinant attenuated Salmonella choleraesuis ΔcrpΔcyaΔasd C78-1(p YA-ORF5-ORF6) was successfully constructed. The recombinant strain was serially passaged in LB solid medium for 60 generations. PCR was performed once every ten generations, and the target band can be amplified, indicating that ORF5-ORF6 fusion gene can be stably inherited in the recombinant strain.