Studies On The Engineered Synechococcus Sp. PCC 7942 That Preparing The Vaccine Anti Shrimp Virus Vaccine

The most serious pathogen in shrimp farming is White Spot Syndrome Virus?WSSV?.However,it has been spreading around the world for more than 20 years.Over the past 10 years,we have studied on the use of transfer vp28 gene cyanobacteria to prepare oral vaccines.The oral vaccine produced by Anabaena sp.PCC 7120 has been tested.In order to further industrialization,it is still necessary to improve the yield of algal cells,the efficiency of VP28 gene expression and the qualitative and quantitative detection on the basis of pharmacodynamics.In this study,Synechococcus sp.PCC 7942 was used to prepare oral vaccine.In improving the above aspects,the following 3 aspects have been made:1.RT-qPCR was used to detect the expression efficiency of vp28 gene in transgenic Synechococcus sp.PCC 7942 at different growth stages.The growth curve of the vp28 gene was measured by the blood cell count board,and the changes of photosynthetic activity of the transgenic Synechococcus sp.PCC 7942 at different temperature,light,pH and salinity were measured by oxygen electrode,and the optimum conditions for its growth were determined.The results showed that the highest expression efficiency of vp28 gene in Synechococcus sp.PCC 7942 was 9.52%,which was three times than that of Anabaena sp.PCC 7120.The optimum harvest time was at logarithmic growth?15d?.The optimum growth conditions for transgenic Synechococcus sp.PCC are:40?,0-0.1 mol/L Nacl,pH 7.5 and light 450?mol·m^-2·s^-1.2.According to the green fluorescence characteristic of eGFP protein stimulated by ultraviolet or blue light,the shuttle plasmid vector pRL489-vp28-egfp expressed simultaneously by vp28-egfp gene was constructed by three parent knot method,and the optimum growth condition of transfer vp28-egfp gene Synechococcus sp.PCC 7942 was determined by the liquid phase oxygen electrode method:the temperature was 40?,and the salinity was 0-0.2 NaCL mol/L;pH was 8;the optimum light intensity range was 400-600?mol·m^-2·s^-1;at the optimum light intensity,the photosynthetic rate of transgenic algae increased by 12.6%compared with that of wild type.The most important thing is to use the transfer vp28 and vp28-egfp gene type Synechococcus sp.PCC 7942 as an immune oral agent.The cumulative death rate of the two experimental groups is 25%,that is,the cumulative survival rate is 75%,the cumulative mortality of the other groups is 100%,and its protective rate for shrimp seedlings is higher than that of the transgenic Anabaena subunit vaccine 7%.3.Through the method of high throughput sequencing of Illumina next seq 500,the transcriptional group of transfer vp28 gene type Synechococcus sp.PCC 7942 was analyzed,mainly by the summary of the quality of the sequencing data,the differential gene expression of the transgenic type Synechococcus sp.PCC 7942,the photosynthesis analysis based on the KEGG pathway,the verification of photosynthesis based on the oxygen electrode and the transcriptional binding.