Expression Of Vp28 In Synechococcus And Purification Of Transgenic Cyanobacteria

It's been over two decades since the WSSV?White Spot Syndrome Virus?was found in 1992.This virus has spared fast in the globe and cause severe harm to the shrimp cultivation and has made great lost to the aquaculture industry globally.However,there has been no commercialized treatment available on the market.Quarantine infected individuals and cultivating strong species are the current measures to deal with the WSSV.During the recent 15 years,one of the structural protein called vp28 was proved to be vital in the infection process of WSSV,and several researchers suggested that subjects get stronger against the WSSV after been feed with vp28 as part of food.Our former research,demonstrated that 70-90% rate of survival in treated shrimp can be obtained after infection of WSSV using genetically modified cyanobacteria as a oral vaccine.Our research project on the oral vaccine made from transgenic cyanobacteria has come to the mid-pilot stage.In this stage,transgenic Anabaena have shown tree constraints affecting the piolt: 1)The wxpression of vp28 was a little low and it was not good enough for fast growing 2)The production conditions in the photoreactor were not manage well so the production wasn't stable 3)Multiple step procedure is esay to get contaminate.This study was tried to give a suitable solution for those issues:1.Constructing a fast growing cyanobacterium by using Synechococcus sp.PCC6803:The Synechococcus sp.PCC6803 have some advantages :1)unicellular and simple structures 2)transformation can be used both natural method and triparental conjugative transfer 3)strong adaptation capability.We synthesis vp28 sequence after searching the Genbank,use the sequence and pRL489 to construct the shuttle vector.Then the vector was amplified in the Escherichia coli and was transfer by triparental conjugative transfer and natural transformation.2.measuring suitable conditions for Synechococcus sp.PCC6803 growth and expression of VP28The exogenous gene makes the genetically modified its' physiology and biochemistry much different from that of the wild type.This study searched the suitable conditions for its growth and the gene expression though both the physiology and biochemistry way.We conclude the best conditions by comparing photosynthetic under different environment factors(illumination intensity 350-400?mol·m^-2·s^-2,temperature 35±5?,pH7.5-8.0).We measured the peak of gene expression by RT-qPCR.3.separation and purification of the cyanobacteria substrainsThe scale cultivation increased the chance of contamination by other bacteria and alge by having more crew and production link.There are some disadvantages of using unpurified substrain:?1?decrease of productivity and bad quality?2?more difficult for further processing?3?side effects of poisonous alga.However,we have not found any publication on the separation and purification of the genetically modified cyanobacteria.There were no significant difference between the genetically modified substrains and the wild type in the same species.So,we need to separate those substrains by special techniques involving physiology,biochemistry and molecular biology.Former research focus on how to separate wild type from the environment,but not separate genetically modified substrains from the wild type.This study has been focusing on the separation and purification of transgenic cyanobacterium,?Anabaena sp.PCC7120?as the subject.To sum up,this study described the methods for constructing the pRL-489-vp28-6803,measuring the suitable condition for production and the vp28 expression,and did some research on separation and purification of the genetically modified cyanobacteria.However,there are still few things left to be done,such as : mass production and drug effect detection of the pRL-489-vp28-6803.