Stability And Safety Of Recombinant Vaccine Against WSSV Developed From Transgenic Cyanobacteria Harbouring Vp28 Gene

White Spot Syndrome Virus(WSSV)is a virus that can cause the explosive death in the shrimp aquaculture.Owe to its fast propagation,wide host range and high fatality rate,WSSV has become one of the serious diseases in the shrimp industry.It has been developed globally a number of drugs,such as recombinant vaccines(DNA vaccines,RNA vaccines,and protein subunit vaccines),and have showed their efficacy at the laboratory.However,up to now there are no effective drugs that can be applied in the shrimp industry.Our research group has used cyanobacteria,a prokaryotic expression system,to express VP28,which based on the important role of major envelope protein VP28 of WSSV in the prevention and treatment of WSSV,and it has been experimentally validated to immunize shrimp effectively.Based on the research progress of our research group over the past decade,this degree article focused on the product stability and safety in the industrialization of the recombinant vaccine,and had the following main results:(1)The stability of vector plasmids was analyzed in light of the loss of target gene during pilot production in this vaccine.The key recombinant vector plasmids p RL-489-vp28 and p GM-T-WSSV-vp28 were maintained the condition of good stability under different antibiotic pressures after transferring into the Escherichia coli(E.coli)TOP10.When p RL-489-vp28 was transformed into the E.coli TOP10 more than 10 h and p GM-T-WSSV-vp28 was transformed over 7h,both of the two kinds of E.coli TOP10 had more than 10% loss rate of the recombinant plasmid rapidly.The OD600 value of the transformed E.coli TOP10 liquid can reach 1.2-2.8 after 10-16 hours of incubation,but the effective bacteria was only between 60% and 85% on average,and the minimum was even less than 55%.The selection pressure in the initial culture was increased to Amp 200 mg/m L and Kana 200 mg/m L,which can effectively increase the relative stability of p GM-T-WSSV-vp28 and p RL-489-vp28 recombinant vector plasmids in transformed E.coli to 85% and 90% rapidly.The transgenic cyanobacteria which containing the recombinant vector plasmid p RL-489-vp28 was subcultured with or without selective pressure,and it was found that the selective pressure(the final concentration of Kana is 200?g/m L)could make its stability still 60.8% after 10 generations.(2)When the vaccine product is operated in the market,the vaccine needs to be keep for a long time until it to be used on the site,so it is necessary to ensure that the active ingredient VP28 protein of the vaccine product is not decomposed or has less decomposition before using in the market.The stability of three consecutive batches of recombinant vaccines at different storage temperatures was explored by using the method of detecting multiple indicators after processing.The using of RT-q PCR,Western Blot and other means can detect the change of the active ingredient VP28 of the recombinant vaccine.At the same time,the biological activity experiment was used to detect the survival rate of challenged Penaeus vannamei after immunization with the recombinant vaccine.The results showed that the VP28 protein content of recombinant vaccine which was stored at-80°C for 6 months was 26.5% higher than that of-20°C and 34.3% higher than that of 4 °C.Oral efficacy experiments showed that the survival rate of the vaccine stored at-80°C was as high as 85% when it was applied to shrimp challenge experiment against WSSV.This proved that the recombinant vaccine can have good stability when it was stored at-80°C for 6 months and promote the growth of shrimp,which is a strong proof of "medicine and food homology".(3)As for an oral agent,the part of the vaccine product could remain in the shrimp larvae when it used,which could cause the release of the transgenic cyanobacteria into the environment,so the transgenic cyanobacteria in the product need to be pretreated.In this study,the freezing-thawing way can be used to ensure that the recombinant vaccine does not lose its active ingredient VP28 under the condition of ensuring its safety,which was also to maintain its stability.After freezing in-20? for 24 h and thawing in room temperature,then repeated tree times,the growth of cyanobacterial cells was lost completely and the cell could not grow on the solid medium.So the freezing-thawing way can control the safe release of the recombinant vaccine against WSSV developed by vp28 gene cyanobacteria and keep its efficacy stability.In summary,we can use 200mg/m L antibiotics to ensure the stability of the recombinant plasmid and avoid the loss of the target gene.And recombinant vaccine can store at-80°C for 6 months to ensure that vaccines are effective.If the transgeniccyanobacterium is freezing-thawing at-20 °C-room temperature before it is made into an oral vaccine product,it can be ensured that the left of product in the shrimp pond will not be released into the environment and not affect the ecological balance.Because of the consideration of operating costs,equipment utilization,and other conditions for commercial production and large-scale using,the recombinant vaccine product can be centrifugally dehydrated into algae mud with freeze-thaw method at-20°C for 3 times(24 hours each time,thawed for 5 minutes),and frozen at-80°C as a guarantee for the safety and stability of recombinant vaccine which against WSSV.Moreover,the use of freeze-thaw method to prevent the release of GMO drug in the environment has not been reported yet,which can provide reference for the safe release and stability of other transgenic cyanobacteria in the environment.The method is easy to operate and has less loss of efficacy.It is an extension of the freezing-thawing way in the scope of application.It also guarantees the stability of recombinant vaccines and provides safety protection for the prevention and treatment of WSSV.