The Different Expression Of Innate Immune Genes After The Challenge Of TC07-2 Type Field IBV Strain In Chickens Immunized With H120 And 4/91 Vaccine

Two commonly used vaccine strains(H120 and 4/91)of Infectious bronchitis virus(IBV)were used to immunize chickens,then a TC07-2 type field strain ZQX firstly isolated in southwestern China wasselectedto challenge the immunized chickens,and the dynamic expression of innate immune related genes were determined.The immune mechanism those two vaccine was analyzed in terms of innate immune,and may provide reference for the reaearch on the immune mechanism of IBV and pathogenic mechanism of TC07-2 type IBV strain.In this paper,one reference gene(ACTB)and five target genes(TLR3,TLR7,MDA5,IFN-?,IL-6)were selected as the targete gene,and method of 2^-??Ctwas used to quantify their expression in fluorescence quantitative RT-PCR.Based on the gene sequences published in Gene Bank,primers were designed and the standard curves of the internal reference gene and the target genes were successfully constructed.Results showed that the amplification efficiency(E)of ACTB,TLR3,TLR7,MDA5,IFN-?,IL-6 of fluorescence quantitative RT-PCR was:1.93,2.00,2.01,1.93,2.09 and 1.93,respectively,and the correlation coefficient(R²)was:1.00,0.99,0.99,0.99,0.99 and 0.99,respectively.The dissolution peaks of all the amplified products are specific single peaks,and the six standard curves constructed have good linear relationship.Sensitivity results showed that the sensitivity of real-time PCR was 100 times greater than that of ordinary PCR.Repeatability results showed that there was good repeatability intra and inter batch within the range of10²?10⁷copies/?L.To sum up,the amplification efficiencies of established standard curves of fluorescent quantitative PCR of a reference gene and five target genes all meet the requirements,the established q RT-PCR have good specificity,high sensitivity,good stability and can be used to for the relative quantitationof targetgenes.To study the dynamic different expression of the innate immune genesafter the challenge of TC07-2 type IBV strain ZQX in chickens immunized by H120 and 4/91,1 day old SPF chicken are divided into A,B,C and D.At 10 dayes of age,chickens in group C and D were inoculated with H120 and 4/91,respectively,chickens in group A and B group were inoculatedwith PBS.At the age of 24days,chickens in group B,C and D were challenged with ZQX,and chickens in group A were inoculted with PBS.Each group was raised separately until 7days post challenge(d.p.c).The clinical symptoms,morbidity,mortality and necropsy of each group were observed and recorded.At 1,3,5 and 7d.p.c,the trachea were collected and q RT-PCR was used to determine the expression of the reference gene and six target genes in the tracheal mucosa of each group,and the differences were analyzed.The results showed that H120 and 4/91 could provide a certain extent of protectionagainst IBV ZQX strain for the aspect of clinical symptoms,morbidity and mortality.The expression of TLR3,TLR7,MDA5,IFN-?and IL-6 were up-regulated in terms of single Innate immune related genes at a certain time point comparing with that the control group,implying thattheyall play a role in fighting against the infection of IBV ZQX.In terms of the peak value of expression of individual genes MDA5,IL-6 and TLR3 stood at the front row.In terms of time when the expression reached the peak,the peak point of the expression of each gene in the H120 group were almost earlier than that of the 4/91immunized group.To sum up,vaccine strains H120 and 4/91 could provide a certain extent of protection against the challenge of ZQX.The selected five target genes play a certain role in the resistance to IBV ZQX infection,but MDA5,IL-6 and TLR3 play a more important role,and the expression of innate immune gene after the challenge of ZQX in chickens immunizes with H120 immunized chickens started more quickly than that of 4/91 immunized chickens.