Establishment And Application Of Bacillus Subtilis System Expressing Cellulase

Traditional Chinese medicine polysaccharides extracted from natural plants are mainly known as having high physiological and pharmacological activities.As a biological response modifier with fewer side effects and safe and non-toxicity,traditional Chinese medicine polysaccharide plays an important role in both immune enhancement and antiviral.However,the traditional method of extracting plant polysaccharides is complex and has a low extraction rate.Resent studies shows that cellulase has a strong destructive effect on the cytoderm of natural plants.In this study,a strain of bacillus subtilis with high expression of cellulase was constructed by genetic engineering technology,which was co-fermented with Astragalus powder to optimize the extraction process and increase the polysaccharide extraction rate.To assess the antiviral effect of fermented Astragalus polysaccharides,using the inhibitory effect of plant polysaccharides on the virus,the Astragalus polysaccharides were combined with the Porcine Reproductive and Respiratory Syndrome Virus（PRRSV）to infect monkey kidney（MARC-145）cells,and it was found that the replication of the virus on the cells was inhibited.In this study,we first constructed the recombinant plasmid producing high cellulase,and further explored the effect of recombinant cellulase-producing bacteria on the extraction rate of Astragalus polysaccharides,providing a simple and efficient extraction method for plant polysaccharides,and it is expected to be widely popularized in animal husbandry.In this study,the Cel9A gene in Bacillus licheniformis was selected as the target gene,and it was ligated to the pHT43 secretory expression vector.The recombinant strain was constructed by CaCl₂ method and induced to express the enzyme.After treatment with the supernatant and precipitate,the SDS-PAGE results showed that there were the same size bands as the target gene was found at 72.36 kDa in the supernatant and precipitate,which indicated that the target gene was successfully expressed.The band of Western Blot at 72.36kDa further indicated that the genewas expressed in B.subtilis.The enzyme production ability was determined by the DNS method and Gram iodine staining method respectively,and it was found that the recombinant bacteria had a much higher ability to produce enzymes than B.licheniformis and Bacillus subtilis.The enzymatic activity was 70U/mL.The recombinant strain expressing a cellulase gene was interacted with Astragalus membranaceus in a fermentation medium,and the content of crude polysaccharide in the medium was determined by a phenol-sulfuric acid method.The results showed that the induced decomposition of the recombinant strain on Astragalus was significantly higher than that of the original B.licheniformis and Bacillus subtilis（P<0.05）,and the extraction rate of the fermentation of Astragalus membranaceus was up to 5.2%.Different concentrations（50mg/mL,100mg/mL,200mg/mL,400mg/mL）of Astragalus membranaceus extract were incubated with diluted TCID₅₀/200 times of PRRSV and inoculated into MARC-145 cells.The TCID₅₀0 was calculated to find that when the virus was incubated with the Astragalus membranaceus extract,the replication of the virus on the cells was weakened and the virus titer was significantly reduced（P<0.05）.Reverse transcription quantitative PCR（RT-qPCR）was performed using RNA from cells of the virus and Astragalus membranaceus extract as a template,and it was found that the viral load was also significantly lowered（P<0.05）.In this study,the recombinant bacteria constructed by genetic engineering technology was applied to the extraction of the fermentation of Astragalus membranaceus.This method saves time compared with the traditional extraction method,reduces the use of chemical reagents,and more importantly,the extraction efficiency is significantly improved,which is industrially the extensive use laid the foundation.Astragalus polysaccharide has inhibitory effects on various viruses,and it acts on PRRSV,which effectively inhibits the replication of PRRSV on cells（P<0.05）,and provides an experimental basis for the treatment of viral infectious diseases in animal husbandry.