Preparation Of Molecular Adjuvant Avian Beta-defensin-5 And Its Application In Enhancement Of Chicks Immune Effect

In recent years,Marek’s disease has caused serious harm to the poultry industry.It’s caused by a herpesvirus known as Marek’s disease virus,whose remarkable characteristics is constantly mutating,decreasing the immune efficacy induced by conventional vaccine.Therefore,it is urgently important to improve the immune efficacy of vaccines.Defensins play a crucial role in the body’s adaptive immune response and immunomodulatory effects.In this study,the chicken β-defensin-5(AvBD5)gene was ligated with the eukaryotic expression vector pcDNA3.1(+)to construct a recombinant expression plasmid.The recombinant pcDNA3.1(+)-AvBD5 was treated as an immune adjuvant emulsified with HVT freeze-dried vaccines toto investigate whether they would improve the immune synergistic functions after injecting chicks.The main contents are as follows:1.Construction of recombinant eukaryotic expression vector pcDNA3.1(+)-AvBD5According to the chicken AvBD5 cDNA sequence(AY621307)in NCBI,primers were designed with restriction enzymes sites Hind III and Kpn I,respectively..The AvBD5 gene was amplified,digested by the double enzymes digestion,and then ligated to the eukaryotic expression vector pcDNA3.1(+).The recombinant pcDNA3.1(+)-AvBD5 was then transformed into E.coli Top10.The single-colony was picked and identified by PCR,double enzymes digestion,and sequences.The results showed that the homologs of amplified gene for AvBD5 shared 100% of identity and similarity with database,and the recombinant plasmid pcDNA3.1(+)-AvBD5 was successfully constructed.2.Chicks immune effect using pcDNA3.1(+)-AvBD5 as adjuvantA large amount of recombinant expression plasmid pcDNA3.1(+)-AvBD5 was extracted as molecular adjuvant.One-day-old chicks were randomly divided into eight groups of 30 each.They were subcutaneously injected with PBS,empty plasmid pcDNA3.1(+),molecular adjuvant,CVI988 vaccine,HVT lyophilized vaccine,and molecular adjuvant combined with HVT lyophilized vaccine,respectively.After that,at the age of 7,14,21,28,and 35 days,five chickens from each group were randomly selected to weight and take blood aseptically.The spleen,thymus,and bursal organs were collected and weighed to calculate the immune organ index of chicks.The collected anticoagulants were processed,and the number of CD3+,CD4+ and CD8+ T lymphocytes were detected by flow cytometry.After the serum was separated from the blood,the contents of serum Ig G antibodies and MD specific antibodies were detected by ELISA method.The results showed that molecular adjuvants could promote the growth of chicken spleen,bursa,and increased the number of CD4+ and CD8+ T lymphocytes in peripheral blood.The combined immunization with HVT vaccine could significantly increase the content of Ig G antibodies in serum and the level of MD specific antibodies.It indicated that the molecular adjuvant had certain immune enhancment ability,which could improve the immune effect of HVT freeze-dried vaccine.