Constrution Of B.Subtilis-vp28Engneeting Strain And The Preliminary Study On Antiviral Mechanism On White Spot Syndrome Virus

White spot syndrome virus (WSSV) can cause shrimp acute, serious disease, resulting in huge economic losses of worldwide shrimp aquaculture, which has become the important pathogen to threaten our country and even world shrimp farming. In this study, The B. subtilis-VP28engineering strain was constructed on the basis of the most abundant WSSV envelope protein VP28as the target antigen protein. The resistance to in vitro gastrointestinal environment of this recombinant strain was assessed to study the most appropriate dosage of industrialization. Then the expression levels of immune genes (proPO, PE, and LGBP) mRNA and phagocytic activity of WSSV were studied. And the strain of survival in the intestinal and distribution in vivo of shrimp after vaccination were detected. A pilot study on potential mechanism of B.subtilis-VP28was also discussd on the methods of affection of non-specific inmune mechanisms and oral delivery. The main research contents and results were as follows:Construction of the Bacillus subtilis strain expressing VP28(B. subtilis-VP28)The VP28gene was inserted into the recombinant plasmid PW980which contain the Quorum sensing promoter Luχ I(from NF003) and wood sugar enzyme poly-peptide signal sequence xynA on the basis of two restriction sites:Pst I and Sal I. The enzyme digestion test and sequencing proved that VP28gene was successfully inserted into the plasmid PW980. Then the recombinant plasmid PW980-VP28was transformed into the host bacteria B. subtilis1A751, which made into competent cells by chemical method. The24h fermentation supernatant of B.subtilis-VP28analysised by SDS-PAGE indicated a distinct band in28KD, consistent with the expected size. This showed the VP28gene was successful secreted to extracellular in the B.subtilis1A751.The resistance assessment of recombinant strain B.subtilis-VP28To evaluate the resistance of recombinant strain B.subtilis-VP28in the gastrointestinal tract environment of shrimp, the counts compared to original inocula were determined after treatment of vegetative cells and spores in the simulated gastric juice and midgut fluid. The result showed that:1) Spores can well-survive and proliferate in the simulated gastric juice with diffrerent pH, which demonstrated the higher resistance than that of vegetative cells;2) Both vegetative cells and spores can resist the simulated midgut fluid of shrimp, and spores are more significant;3) The percentage of surviving spores was93.9%after treatment at the temperature of80℃, while the vegetative cells were almost inactivated at this condition. The conclusion was that the B.subtilis-VP28spores with higher resistance against the simulated gastrointestinal condition and high temperature can survive and colonize in vivo of shrimp. These key features make this recombinant strain B.subtilis-Vp28spores as the ideal dosage form to delivery VP28protein to extreme enviroments such as gastrointestinal.The immune genes mRNA expression and haemocyte phagocytic activity of shrimp after shrimps vaccinated recombinant strain B. subtilis-VF28The shrimp were vaccinated with recombinant strain B.subtilis-WP2S fermentation supernatant (mainly VP28), B.subtilis, and NaCl group as control, respectively. SYBR Green I qRT-PCR was applied to detect the immune genes of proPO, PE and LGBP mRNA expression levels. WSSV virions were labeled by FITC for the detection of haemocyte phagocytic activity. The results showed that B.subtilis-VP28can significantly affect the proPO, PE and LGBP mRNA expression levels and haemocyte phagocytic activity of WSSV. B.subtilis compared with B.subtilis-VP28fermentation supernatant had a little influence on the shrimp immunity. It was explained that recombinant strain B.subtilis-VP28can significantly affect the shrimp's non-specific immune response, and the B.subtilis itself as the probiotic also played a certain role on the shrimp'immunity.Survival of B.subtilis-VP28in the intestine of shirmp and the distribution in vivoAccording to the antigen specfic of VP28and the heat-resistant of spores, shrimp were orally delivered a count of spores and then detected its survival in the intestine by spore counting. At the same time, the frozen sections of gut, glands, heart and muscle were made for immunoflurescence staining combined with PCR and spore count method to evaluate the distribution of B.subtilis-VP28in the shrimp. The results showed that the spores were extremely robust with most colonization and survival in the shrimp intestine. Three methods all showed that B. subtilis-VP28can be detected in the intestine, stomach and glands except for the muscle.PCR method and immunofluorescence staining method detected the signals in the heart after immuned7d.This may be the circulation and penetrate of haemocyte promoting the B.subtilis-VP28protein in shirmp.