Establish The Transformation System Of Cordyceps Mycelium And Transforming The Gene Of PRRS Vaccines

Porcine reproductive and respiratory syndrome syndrome(PRRS), also known as blue ear pig disease, is a serious infectious disease caused by Porcine reproductive and respiratory syndrome virus(PRRSV), it not only affects the swine industry and food security seriously, but also causes serious economic losses to the swine industry all over the world.At present, the effective and important means to prevent PRRS is vaccination, in which is a new type of genetic engineering vaccine that is not only able to achive the immune effect,but also can reduce human and material resources. Therefore, Using Cordyceps militaris as a receptor system, the choice of some fungi which contains pharnaceutical and bioactive components and the development of PRRS edible vaccine have a good application prospect. C.militaris is a kind of representative entomopathogenic fungi used for medicinal purposes in eastern Asian countries. According to traditional Chinese and modern medical research, cordycepin has effects of anti-tumor, anti-inflammatory and immune regulation. As rich and active ingredients of edible and medicinal fungi, the method of commercial production of C.militaris method has been established, but the safe and efficient transformation system is still in the initial stage. In this paper, we used C.militaris mycelium as a biological reaction system first time, established a stable and efficient transformation system of C.militaris; and successfully cloned the PRRS vaccine gene ORF3 and ORF5 which were cloned into the vector p CB130 NG. The protoplast of C.militaris was transformated by PEG, and the mycelium ORF3 gene and ORF5 gene were obtained respectively. The research results are as follows:1. The optimum conditions of extracting the protoplast of C.militaris:mycelium mat age is 4 day, the osmotic pressure is 0.8 mol/L p H of mannitol is 4.5, and 1.5% snail enzyme and lywallzyme,were enzymolysis at 34 ? for4 h. FDA staining was used to detect the activity of the protoplast, and the activity can be more than 90% with normal shape and same size.2. Through screening the regeneration medium for the protoplast of C.militaris, the optimal regeneration medium was determined as PDA medium containing 0.8 mol/L mannitol.3. The concentration of hyg was determined as 500 mg/Lby the experiment of the concentration gradient.4. The p CAMBIA1302 vector with green fluorescent protein(GFP) gene was used to establish the transformation system of C.militaris, and determine the optimal transformation conditions that the number of protoplast is 107, 30 ?g plasmid, 25%PEG and ice bath for 10 minutes.5. Based on literature review, the main structural protein of PRRSV is determined to be GP3 and GP5, and the coding gene is ORF3 and ORF5. The two genes were connected to the fungal specific expression vector p CB130 NG, and constructed the expression vector p CB130 NG – ORF3-ORF5 after sequence design6. The gene ORF3 and ORF5 of the protein GP3 and GP5 were successfully cloned by enzyme digestion an ligation, which constructed the expression vector p CB130NG-ORF3 and p CB130NG-ORF5.7. The expression vector p CB130NG-ORF3, p CB130NG-ORF5, p CB130NG-ORF3-ORF5 was respectively used to transformate the protoplas of C.militaris by PEG, and the transformants were proved preliminarily that the vaccine gene of PRRSV was successfully transformated in C.militaris by PCR.