Research On The Preparation Of Linear Influenza DNA Vaccine By Large Scale PCR And Its Efficacy

Influenza is an infectious disease caused by an influenza virus of typ A,and has been spread around the world.The most effective protection against influenza is vaccination.While,due to the viruses' ability to change its envelope glycoprotein hemagglutinin(HA),neuraminidase(NA)via shift or drift,permitting influenza to escape the protection induced by natural immunity or current vaccines,which require the development of new vaccines nearly every season.It has been clear that successful prevention of an outbreak of a highly virulent influenza strain will need fast manufacture of large amount of vaccines.While,manufacture and development of tradtional influenza vaccines require the use of outdated technologies that have attested slow and unreliable and are therefore impossible to meet the challenges of a potentially rapidly spreading and changing pandemic.It is reported that vaccination with influenza A HA-expressing LEC DNA could protect BALB/c mice against a lethal dose of influenza A.Efficacy data as well as easy to manufacture and formulation support further investigation of LEC DNA as a promising alternative of traditional vaccines.At present,PCR is the main method of producing linear DNA vaccine.However,the scale and cost of conventional PCR has become the main bottleneck in the large-scale preparation of linear DNA vaccines.The main factor causing the high cost of PCR is the high cost of the thermostable DNA polymerases.To overcome this problem,we tried to secretory expression the DNA polymerase with Pichia pastoris to reduce the cost.The DNA coding sequences of KOD and Sso7d were optimized to match the codon usage preference of Pichia pastoris and Sso7d was fused to the C-terminus of KOD.The resulting novel gene was then cloned into a pHBM905A vector and introduced into P.pastoris GS115 for secretory expression.The expression of RKOD reached a maximum level at 120 h,with a protein concentration of approximately 250 mg/LL,the enzymatic activities of purified RKOD was approximately 19,384 U/mg.In this study,thermostable DNA polymerase was successfully secretory expressed with Pichia pastoris expression system,simplifying the purification steps,saving the cost of production.And on this basis,we have explored a large scale PCR strategy based on a simple water bath equipment.In this study we successfully reached 10 mL,20 mL,50 mL and 100 mL PCR.And a large number of HIN1 linear DNA vaccine were prepared via the large-scale PCR technology in this research.In order to verify the protective effect of linear DNA vaccine prepared by large-scale PCR.BALB/c mice were immunized and challenged with H INI virus.The results indicate that,the linear influenza vaccine prepared by large-scale PCR in this study can effectively protect mice against the virus challenge.And the PTO-LEC-HA vaccine afford full protection against challenge in a mouse model.In conclusion,we have successfully established a method for the production of linear DNA vaccines by large-scale PCR.This research bring a new strategy to produce DNA vaccine massively and effectively to respond rapidly to newly infectious diseases.