Construction And Identification Of Recombinant Adenovirus Expressing S1 Gene Of IBV And TM-1 Gene Of MG

Avian infectious bronchitis?IB?is an acute and highly contagious infectious respiratory disease caused by avian infectious bronchitis virus?IBV?,which is one of major infectious respiratory diseases in word poultry.As a major immunogenic protective protein of IBV,there have been many reports about using S1 protein to immune chickens and achieve good results.Chronic respiratory disease?CRD?is a contagious and chronic infectious respiratory disease caused by Mycoplasma Gallisepticum?MG?,whichmainly cause decreased egg production and quality,declining in fodder conversion ratios,and the disease brings huge economic losses.TM-1 protein of MG is related to adhere to host cell,which has been shown in capable of inducing protective immunity in chickens.At present,we haven't found the related reports about bivalent genetic vaccine using S1 gene and TM-1 gene recombinat adenovirus.In this research,we amplified target genes from IBV H52 strain and MG S6 strain using RT-PCR and PCR respectively,then cloned the target genes into pMD19-T vector by T-A cloning,named pMD-S1 and pMD-TM-1.After identified by digestion and sequencing,we used T4 ligase to link the target genes with pDC₃₁₅-EGFP,and obtained recombinant adenovirusshuttleplasmidpDC₃₁₅-S1-EGFP,pDC₃₁₅-TM-1-EGFPand pDC₃₁₅-S1-TM-1-EGFP respectively.HEK293 cells were cotransfected with the constructed recombinant shuttle plasmid and pBH by lipofection.After tested by western blot,we constructed recombinant adenoviruses expressing S1 gene and TM-1 gene,which were named pBH-S1-EGFP,pBH-TM-1-EGFP,pBH-S1-TM-1-EGFP respectively.We purified by Vivapure AdenoPACK and determined with TCID₅₀0 method,and the titers of pBH-S1-EGFP,pBH-TM-1-EGFP,pBH-S1-TM-1-EGFP and pBH-TM-1-EGFP were 10¹0.8750.875 TCID₅₀/mL,10¹1.7501.750 TCID₅₀/mL,10¹0.6250.625 TCID₅₀/mL and 10¹2.2502.250 TCID₅₀/mL respectively,which is enough for follow-up animal experiment.By measuring the titer of recombinant adenovirus at different time,we drawn its a step growth curve,and the curve showed that growth characteristic of the recombinant adenoviruses are basically consistent with the adenovirus.The P4,P8,P10,P15 recombinant adenovirus contained S1 gene and TM-1 gene were detected by PCR,the results showed a good genetic stability of the recombinant adenovirus,which lay the reaearch basis for the further animal experiment of the bivalent genetic vaccine.This study aims to construct the recombinant adenovirus expressing IBV S1 gene and MG TM-1 gene,and illuminates its growth characteristic and genetic stability,which lay the foundation for research on bivalent genetic vector vaccine of avian infectious bronchitis and chronic respiratory disease.