Cloning And Sequencing Of Gallus Domesticus Brisson IL-16 And Studyon Its Adjuvants Immunocity On Avian Infectious Bronchitis DNA Vaccine

Gallus domesticus Brisson IL-16 was amplified by RT-PCR from spleen tissue total RNAstimulated with ConA. Then cloned into PMD18-T vector and sequenced. The resultshowed that the open reading frame of IL-16 was 1824bp,encoding 608 amino acids. Sharinghomology with the Gallus IL-16 gene in Genebank,we Found that the nucleotide sequenceshared 99.7％homology with the ChIL-16 gene of Xu,X.And the nucleotide sequence shared99.5％and 99.2％homology with the ChIL-16 gene of Min,W and Sayed,A.A respectively.Wefirstly cloned ChIL-16 gene in domestic. The cloned gene of chIL-16 in our experimentenriched the gene banks and provided the material for further study and lay the foundation forfurther study.Then digested the gene with BamHⅠand ECORⅤ, inserted into pcDNA3.1(+) whichwas also digested with BamHⅠand ECORⅤ. The eukaryatic expression vector of chickeninterleukin-16(pDNAIL-16) was constructed and identified. We simultaneously immunizedchicken with IBV DNA vaccine and pDNAIL-16 respectively or together.The immunologicalenhancing effects of molecular adjuvants IL-16 to IBV DNA vaccine was investigated. 140SPF chicken (1-day-old) were divided into 7 groups(PBS group,pcDNA3.1 group,IBV DNAgroup, pDNAIL-16+IBV DNA16 group,inactive IBV vaccine group,attenuated group).Withindirect ELISA admeasurement, pDNAIL-16+IBV DNA16 group induced the highest level ofspecific antibody within 35 days.Antibody level of pDNAIL-16+IBV DNA group comparingwith inactive IBV vaccine group and attenuated group was discovered extremely significantdifference after14 the 14 day(p＜0.01), pDNAIL-16+IBV DNA group compared with allother groups was also discovered extremely significant difference after the 21 day(p＜0.01).The result of detected quantities of CD4+,CD8+ and CD3+T cell subtypes indicated that pDNAIL-16+IBV DNA vaccine extraordinarily raise their amount,especially promotequantities of CD4+ T cell subtypes in prophase of immunity.Quantities of CD4+,CD8+ andCD3+T cell subtypes can reach the most after chicken immunized with pDNAIL-16+IBVDNA vaccine 14 days later. Quantities of CD4+,CD8+ and CD3+T cell subtypes of othergroups can resch the most after immunized 21 days later. CD3+T cell quantity thatpDNAIL-16+IBV DNA was discovered significant difference comparing with DNA vaccinegroup in 21day (P＜0.05), and was discovered extremely significant difference comparingwith other groups in 21day (P＜0.01).By the experiment of injection IBV to Gallus domesticus Brisson pDNAIL-16+IBV DNAgroup its protection ratio (90％) obviously was higher than the independent IBV DNA group(85％), and the same as attenuated IBV DNA vaccine's protection ratio,but lower thaninactived IBV vaccine's protection ratio.The study also showed that IBV DNA vaccine mainly has effect in mid-stage of immunity.Molecular adjuvant pDNAIL-16 can be critical in early-stage of immunity.Especially itquickly enhanced cell-mediated immunity。Co-administration with IBV DNA vaccine andpDNAIL-16 could sustain high level of immunity in all mid-stage of immunity.The resultindicated that pDNAIL-16 could enhance immunifaction of IBV DNA vaccine.And it can be agood adjuvant in immune enhancement.