1.Prokaryotic Expression of Bm86 Gene of Boophilus microplus and Optimization of the Expression Condition
Miling MA ; Guiquan GUAN ; Youquan LI ; Aihong LIU ; Qiaoyun REN ; Qingli NIU ; Hong YIN ; Jianxun LUO
Chinese Journal of Parasitology and Parasitic Diseases 1997;0(06):-
A pair of specific primers was designed based on the reported Bm86 gene of Boophilus microplus,the Bm86 gene was cloned by PCR using the plasmid pMD18-T-Bm86 as templates,and subcloned into the prokaryotic plasmid pGEX-4T-1.The recombined plasmid was transformed into E.coli BL21(DE3) and followed by expression of the protein induced by different concentration of IPTG for different time.SDS-PAGE showed that the recombinant plasmid pGEX-4T-1/Bm86 expressed a fusion protein Bm86-GST(Mr 94 000) after being induced with IPTG.High level expre-ssion of Bm86-GST was found at 1 mmol/L IPTG condition after incubation for 8 h at 37 ℃,and the expression level of the recom-binant Bm86-GST reached up to 29% of total E.coli proteins.Western-blotting analysis showed that the recombinant Bm86-GST was recognized by the rabbit anti-B.microplus positive serum.
2.The E248R protein of African swine fever virus inhibits the cGAS-STING-mediated innate immunity.
Yinguang LIU ; Wenping YANG ; Yuan WEN ; Qingli NIU ; Jifei YANG ; Guiquan GUAN ; Hong YIN ; Haixue ZHENG ; Dan LI ; Zhijie LIU
Chinese Journal of Biotechnology 2022;38(5):1837-1846
We researched the mechanism of African swine fever virus (ASFV) protein E248R in regulating the cGAS-STING pathway. First, we verified via the dual-luciferase reporter assay system that E248R protein inhibited the secretion of IFN-β induced by cGAS-STING or HT-DNA in a dose-dependent manner. The relative quantitative PCR analysis indicated that the overexpression of E248R inhibited HT-DNA-induced transcription of IFN-b1, RANTES, IL-6, and TNF-α in PK-15 cells. Next, we found that E248R interacted with STING by co-immunoprecipitation assay and laser confocal microscopy. Finally, we demonstrated that E248R inhibited the expression of STING protein by using Western blotting. We demonstrated for the first time that the E248R protein of ASFV suppressed the host innate immune response via inhibiting STING expression. The results are pivotal in extending the understanding of the ASFV immune escape and can guide the design of vaccines against ASFV.
African Swine Fever Virus/genetics*
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Animals
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DNA
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Immunity, Innate
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Nucleotidyltransferases/metabolism*
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Signal Transduction
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Swine