Functional Microorganism Analysis Based On Theillumina-based Sequencing Method And Its Application In Thedevelopment Of Shrimp White Spot Syndrome Virus Vector Vaccine

As an important part of the natural ecosystem,microorganisms are ubiquitous in animals,plants,and other natural environments.They are important for maintaining the ecological balance of the natural environment and the physiological functions of the host.Therefore,the diversity and function of environmental microbes have become a research hotspot in microbiology.Due to the limitation of conventional culture techniques,less than 1% of the microorganisms can be used for isolation and cultivation in the various environments of natural ecosystem at present.Therefore,the method of molecular biology that does not rely on cultivation has been widely used in environmental microbiology research.However,at present,the research focuses on these methods are mostly limited to the analysis of the diversity of environmental microbes,and few relative excavations and analysis of specific functional microbes directly related to the environment or host function.Therefore,in order to better explore the application effects of Illumina-based sequencing method in functional microbial excavation analysis.This article mainly use the Illumina-based sequencing method to analyze the diversity of the microorganisms and functional flora by sequencing the bacterial 16 S rRNA gene V3-V4 hypervariable region of the goat intestinal probiotic group and the fungal rRNA gene ITS2 region of Ophiocordyceps sinensis.Then,based on the analysis results of intestinal flora of shrimp and the research status of white spot syndrome virus(WSSV),the dominant flora in shrimp intestinal was isolated.At the same time,the appropriate vector promoter was selected.The isolated Enterobacteriaceae.spp was used as a carrier strain to construct the vector vaccine.The main results are as follows:(1)The Illumina-based sequencing method with different specific primers for Bacillus,Clostridium,and Bifidobacterium were applied to analyze the diversity of the genera in goat feces.The results suggest that the average number of different Bacillus,Clostridium,and Bifidobacterium OTUs(operational taxonomic units)at the 97% similarity level ranged from 1922 to 63172.The coverage index values of Bacillus,Clostridium,and Bifidobacterium calculated from the bacterial OTUs were 0.89,0.99,and1.00 respectively.The most genera of Bacillus(37.90%),Clostridium(52.50%),and Bifidobacterium(99.32%)were detected in goat feces by the Illumina-based sequencing with the specific primers of the generarespectively.(2)Sequencing analysis of the fungi in Ophiocordyceps sinensis using Illumina-based sequencing method shows that a large number of target fungal sequences were sequenced in the sample of worms and subunits of Ophiocordyceps sinensis.The number of fungal OTUs(operational taxonomic units)at the 97% similarity level ranged from 75 to 187.The coverage index values were 1.00.There are a large number of common fungi and their ownflora in the worms and subunits.Ophiocordyceps(61.51%.5.30%)and Mortierella(26.86%.5.91%)are the dominant communities,Tolypocladium,Paecilomyces and Metarhizium are also detected and most of them are common flora.(3)According to the analysis results of the microbial populations in different samples by using Illumina-based sequencing method,and the analysis results ofthe Enterobacteriaceae(85%)ofGammaproteobacteria is the dominant intestinal florain all growth stages of shrimp.In this experiment,the intestinal bacteria were isolated and cultured.The results suggest that the Enterobacteriaceae(81%)is the dominant species.(4)In order to screen the suitable vector promoters,the express vectors was constructed by using different ferric uptake regulat promoters(fur promoter)and phage PhiX174 lysis gene E.The results suggest that the construction of bacterial ghosts vaccines and the screening of vector promoters in this experiment is not good,and it is necessary to further screen thesuitable promoters that influence the expression of specific virulence proteins.(5)Combined withthe analysis results of intestinal flora of shrimp and separation of the dominant intestinal flora of shrimps and the screening results of appropriate promoters,the constitutive promoter J23100 and the N-terminal gene sequence of ice nucleation proteininaK-N of P.syringae and the white spot syndrome virus(WSSV)envelope protein VP28 gene sequence were recombined to construct the expression vector pJIVP28.The dominant strain of Enterobacteriaceae isolated from the intestine of shrimp was used to construct the WSSV vector vaccine.The results suggest that the recombinant expression vector was constructed successfully and the WSSVVP28 protein expression detected by ELISA has a certain activity and is also expressed stably in the host bacteria.In summary,the results of this paper shows that there are a large number of functional microorganisms in the intestinal tract of animals,and the use of specific sequencing analysis methods will facilitate its in-depth excavation and development.The natural resources of Ophiocordyceps sinensis are depleted and it is difficult to plant artificially.In the previous studies,only one species of fungi was used to plant the Ophiocordyceps sinensis militaris but failed.By comparing Cordyceps(99%)sequencing results in Cordyceps militaris,we can conclude that Ophiocordyceps sinensis may be formed by both species.This is of great significance for the isolation,culture and artificial cultivation of Ophiocordyceps sinensis.Shrimp White spot syndrome sirus(WSSV)is a kind of enterovirus.By analyzing the changes of gut microbiota in different growth stages of shrimp,the dominant flora was isolated and cultured,and used as a vector strain to construct a related vector vaccine.It has great significance for the development of aquatic aquaculture.