The Application Of Cordyceps Militaris For Development Of Edible Vaccine Of Porcine Rotavirus Vaccine

Porcine rotavirus(PoRV)can cause viral diarrhea in piglets,with 60% infection rate and 20% mortality rates,resulting in annual economic losses of billions of dollars.Rotavirus can still infect the body in harsh environments,no effective treatment after infection for effective treatment.The best way to prevent the occurrence of rotavirus disease is vaccination.PoRV structural proteins VP7 and VP8* can induce antibodies to the body and serve as candidate proteins for vaccine development.In recent years,the development of genetic engineering leaps and bounds,and the fungi is used as a carrier.The production of medicinal protein or peptide has the advantages of plant genetic engineering,and can achieve large-scale industrial production.However,the study of porcine rotavirus expression in Cordyceps militaris has not been reported.Cordyceps(Cordyceps militaris),is also known as Cordyceps sinensis.Its active ingredients are Cordyceps,Cordyceps,polysaccharides,etc.,which can inhibit the tumor,lower blood sugar,enhance immunity and has other effects.In recent years,the development of genetic engineering by leaps and bounds,the use of fungi as a carrier,the production of medicinal proteins or peptides are widely concerned.Cordyceps militaris as a medicinal fungus,itself has a variety of pharmacological activity,widely used.With the whole genome of Cordyceps militaris,its molecular biology and genetics research will be more in-depth.In this study,Cordyceps militaris was used as the receptor system to express porcine rotavirus protein.The aim of this study was to obtain transgenic Cordyceps militaris,which could support the production of drug protein in Cordyceps militaris.The development of swine rotavirus Feeding the vaccine,both to produce piglets immune,but also enhance the pig's immunity,greatly improve the survival rate of piglets,with high practical value and economic value.The results of this study are as follows:1.In order to obtain a porcine rotavirus antigen protein gene suitable for Cordyceps militaris expression,the amino acid sequences of porcine rotavirus VP7 and VP8* proteins were screened from NCBI and optimized by using Cordyceps militaris.The two proteins were inserted into the 2A-linked peptide.Six His tags were fused before and after the sequence to make separation and purification as well as the synthesis of gene sequences more convenient.2.The VP8 and VP7 gene of porcine rotavirus were amplified by PCR using VP7-VP8 * fusion gene as template.The gene was cloned into the fungal expression vector pCB130 to construct a recombinant plasmid.The specific expression vectors pCB130-VP7-VP8*,pCB130-VP7 and pCB130-VP8* were successfully constructed.The gene was cloned into prokaryotic expression vector pET30 a to construct recombinant plasmid.The prokaryotic expression vectors pET30a-VP7-VP8*,pET30a-VP7 and pET30a-VP8* were successfully constructed.3.The prokaryotic expression plasmid was transformed into E.coli BL21 competent state.Screening positive colonies by PCR.The VP7-VP8*,VP7 and VP8* proteins were expressed by IPTG.SDS-PAGE was used to detect the expression of protein,and the bands were detected at the size of the target protein.The results showed protein expression.The protein was detected by Western Blot,and the bands were detected at the size of the target protein.The results showed that the recombinant protein could bind to the antibody and had good immunogenicity.4.Successful Establishment of Agrobacterium-mediated Transformation of Cordyceps Militaris Protoplast System.In this study,we found that the ratio of compound enzyme prepared by protoplast of Cordyceps militaris was snail enzyme: lysozyme = 1: 1;the optimum enzymolysis time was 4 h and the morula was 4 d at 34 °C.5.Fungi-specific expression plasmids were transformed into Agrobacterium tumefaciens by freeze-thawing method.Transformation of Cordyceps Militaris by Agrobacterium-mediated Transformation.A total of 14 strains of transgenic VP7-VP8* gene were used to isolate Cordyceps militaris,19 strains of VP7 gene cordyceps militaris and 8 strains of VP8* gene cordyceps militaris.Transgenic Cordyceps Militaris Strains were screened by PCR.After screening,14 strains of VP7-VP8* gene were killed,5 strains of VP7 gene cordyceps militaris and 5 strains of VP8* gene cordyceps militaris.The expression of protein was detected by Western Blot.Five strains of Cordyceps militaris were identified as positive,and the positive rate was 28%.DNA detection and protein level detection of transgenic Cordyceps militaris showed that the gene had been integrated into the Cordyceps militaris genome and expressed.