| Marek’s disease is a highly contagious immunosuppressive tumor disease.The pathogen is Marek’s disease virus of the Herpesviridae family,which has a serious impact on the poultry industry.At present,the early prevention of the disease is mainly vaccinated,but the effect is limited.Because the anti-viral effect of Chinese herbal medicine in our country is real and there is no residue,many researchers have now focused on the research of Chinese medicine anti-viral.There are more and more researches on anti-herpes virus,and there is no lack of other traditional Chinese medicines on Marek’s disease virus,and the research of Radix Isatidis polysaccharide on Marek’s disease virus has not been reported.Therefore,in this experiment,Radix isatidis polysaccharide was used as the test drug to study its effect on the replication of Marek’s disease virus Md5 BAC on primary chicken embryo fibroblasts in vitro.1.Research purpose:In this experiment,the effects of three administration routes of Isatis indigotica polysaccharide on the replication of Marek’s disease virus super-virulent Md5 BAC on primary chicken embryo fibroblasts were mainly tested in vitro,and the anti-virus of IRPS was initially explored at the level of DNA,mRNA and protein.The mechanism of action provides a new and reliable method for the prevention and treatment of Marek’s disease in chickens.At the same time,it is hoped that the exploration of this experiment will lay a theoretical foundation for the antiviral effect of Radix Isatidis polysaccharide.2.Research methods:(1)The cell maintenance solution for IRPS is diluted to a series of concentrations.After the CEF grows into a single layer,add it to the culture plate.By measuring the cell OD value,the cytopathic observation method(CPE)and the method of counting tiypan blue cells,Evaluate the effect of different concentrations of IRPS on the vitality of CEF,so as to determine the maximum safe concentration of IRPS on CEF cells.Observing the apoptosis of CEF by Hoechst 33258 fluorescent staining also confirmed the maximum safe concentration of IRPS on CEF cells.(2)Dilute the Md5 BAC virus 10 times with the cell maintenance solution to a serial concentration,add it to a 96-well cell culture plate,and use the Reed-Muench method to calculate the half cell infection amount(TCID50)of Md5 BAC.On this basis,using CEF as the host cell,by measuring the cell OD value from IRPS,the three routes of administration of Md5 BAC virus(first adding virus and then polysaccharide,first polysaccharide and then virus,polysaccharide and virus mixed feeling after Add to cultured CEF cells)to evaluate the effect of IRPS on the ability of the virus to infect cells.(3)Using real-time fluorescent quantitative PCR technology to detect the DNA level of 320 μg/mL and 640 μg/mL IRPS on Md5 BAC virus gene ICP4 copies at 24,72,and 168 hours by mixing the virus and drugs for 4 hours and then adding them to the cells.The influence of the number of cytokines on the expression of cytokines caspase3,IL-6 and IFN-β was detected at the mRNA level of 320 μg/mL and 640 μg/mL IRPS;320μg/mL and 640 μg/mL IRPS were detected by indirect immunofluorescence technology Effect on the fluorescent plaque area of Md5 BAC virus gB protein.The effect of 320 μg/mL and 640 μg/mL IRPS on gB protein expression was detected by Western Bolt technology.3.The research results are as follows:(1)The maximum safe concentration of IRPS for CEF is 640 μg/mL.(2)The Md5 BAC’s half-cell infection(TCID50)for CEF is 10-4.3/0.1 mL,that is,13.7×104 pfu/mL.(3)The maximum proliferation inhibition rate of IRPS on Md5 BAC was 36%,infection blocking was 29%,and direct inactivation was 53%.(4)At the DNA level,the copy numbers of IRPS at 320 μg/mL and 640 μg/mL ICP4 are both lower than those of the virus control group,but the difference is not significant(p>0.05).The expression of the apoptotic gene caspase-3 at the mRN A level The expression of caspase-3 on Day7 was significantly higher than that of the virus control group,and the difference was extremely significant(p<0.01)The expression of inflammatory factor IL-6 gradually decreased with the increase of IRPS concentration.IL-6 was significantly lower than the virus control group when the IRPS concentration of Day1 was 640 μg/mL,the difference was significant(p<0.05).(5)At 320 μg/mL IRPS and 640 μg/mL IRPS,the area of fluorescent plaques of gB protein was significantly reduced compared with the virus control group(P<0.01).(6)At 320 μg/mL IRPS and 640μg/mL IRPS,the expression of gB protein was significantly reduced compared with the virus control group(P<0.01).4.Conclusion:Radix Isatidis polysaccharide can inhibit MDV by inhibiting the copy number of viral genes,affecting protein expression and regulating cytokines,among which direct killing effect is the best. |
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