Construction Of Recombinant Adenovirus Fusion Expressing The TM-1 Protein Of MG And The GD Gene Of ILTV And Its Research On Immunological Efficacy

Chronic Respiratory Disease(CRD),caused by Mycoplasma gallisepticum(MG),is a chronic,contagious respiratory disease in chickens.The clinical signs of CRD is characterized by difficulty breathing,severe coughing,a lowered immunity and can predispose a secondary viral or bacterial infections.Infection persistently of MG can cause the chicken group production performance decreasing and body abandoned,which would bring a huge economy loss for the poultry industry in domestic and overseas.TM-1 protein of MG is hemolysin membrane protein that is related to adhere to host cell.And the protein has the characteristic of high conservative,low variance,which has been shown in capable of inducing protective immunity in chickens.Infectious Laryngotracheitis(ILT)is an acute and highly contagious respiratory disease in chickens,which caused by the Infectious Laryngotracheitis Virus(ILTV).The disease can lead to death and egg production decreases.Infection of ILTV brings huge economic losses around the world and had been one of the major diseases that endanger the poultry industry.As a major immunoprotective protein inducing immune response in chickens,gD glycoprotein plays an important role in the process of ILTV attaching and enter into the target cells.In this research,after identified by PCR,double digestion and sequencing analysis,pMD-TM-1 and pMD-gD were treated with double digestion in order to acquire the terget genes of TM-1gene and gD gene.And T4 DNA ligase was used to link the target genes into shuttle vector to construct pDC315-TM-1,pDC315-gD and pDC315-TM-1-gD respectively.After identified by PCR,double digestion and sequencing analysis.Then these recombinant shuttle vectors contrusted successfully were co-transfected into HKE293 cells with constructed pBHGlox(delta)E1,3cre simultaneously in the help of LipoFiterTMlipofection,making vectors homologous reorganizations in eukaryotic cells.After detecting by fluorescence detection,RT-PCR and Western blotting,it was verified that recombinant adenoviruses were constructed successfully.After infected these recombinant adenoviruses,total RNA of infected HEK293 cells were collected and the results of RT-PCR showed that the gene of TM-1 and gD were expressed efficiently.Simultaneously,total protein of infected HEK293 cells were collected and the results of Western blotting showed that the protein ofTM-1 and gD were expressed efficiently.Vivapure AdenoPACKTM was used to purify these viruses in prepare for the animal experiment.TCID50 detected viruses titers: 109.5TCID50/mL?109.625TCID50/mL?109.25TCID50/mLand 1010.125TCID50/mL respectively,which were enough for follow-up animal experiment.7d SPF chickens were conducted with immune procedure in7 d intervals.The chickens immuned did not showed typical clinical symptom.And bodyweight of immuned chickens had no significant difference comparing the control group Taking the blood sampling in 7d,14 d,21d and 28 d post immunization,indirect ELISA showed that there were not significant difference between the groups of pBH-TM-1,pBH-gD(p>0.05).The level of MG antibody induced by the pBH-TM-1-gD was significantly lower than that of the conventional vaccine(p<0.01).However the level of ILTV antibody induced by the pBH-TM-1-gD was no difference with that of conventional vaccine(p>0.05).Thymus index analysis showed that thymus index of pBH-TM-1,pBH-gD,pBH-TM-1-gD and the conventional vaccine group were significantly increased(p<0.05)comparing with control,and between pBH-TM-1,pBH-gD,pBH-TM-1-gD and the conventional vaccine group no difference was found.In this research,TM-1 gene of MG and gD gene of ILTV were fusion reorganized and were successfully constructed the recombinant adenovirus pBH-TM-1-gD,which can be used for the fusion TM-1-MG protein expression of eukaryotic system.On the basis of the comparison with the conventional vaccine,we discussed its immunological efficacy.This research provide a theoretical basis for the development of new poultry respiratory tract vaccine and can also be a reference for the development of two kinds of avian carcinogenic infectious diseases.Simultaneously,this research can provide a theoretical basis for the new poultry respiratory vaccine.