非洲猪瘟病毒MGF505-3R蛋白的原核表达研究

为获得非洲猪瘟病毒(African swine fever virus,ASFV)体外诱导表达的MGF505-3R蛋白。实验依据GenBank中报道的MGF505-3R基因序列设计引物,引入His标签,以ASFV基因全序列为模板扩增目的基因。将目的基因克隆至原核表达载体pGEX-6p-1,并转入大肠杆菌Transetta(DE3)中。用1 mmol/L异丙基β-D-硫代半乳糖苷(IPTG)诱导表达菌体,分别取上清液和沉淀进行十二烷基硫酸钠-聚丙烯酰胺(SDS-PAGE)凝胶电泳分析。将表达的蛋白经过Ni-NTA琼脂糖亲和层析纯化。免疫印迹实验(Western-blot)分析蛋白的活性。克隆得到的MGF505-3R基因片段长度为843 bp,与原序列一致。蛋白在沉淀中以包涵体的形式表达,相对分子质量约为53 ku。纯化后可得到纯度较高的MGF505-3R重组蛋白。免疫印迹实验结果呈阳性。MGF505-3R蛋白可以在大肠杆菌Transetta(DE3)中表达并纯化,蛋白具有良好的表达活性。近几年,多基因家族(Multigene Families,MGFs)基因缺失疫苗被认为是目前ASF疫苗研制的主要方向,本研究获得的MGF505-3R重组蛋白将为ASFV MGF505-3R蛋白的研究提供基础,也为基因缺失疫苗相关免疫学方面的检测提供前期技术储备。

非洲猪瘟病毒MGF505-5R蛋白表达与纯化

为了获得具有生物学活性的非洲猪瘟病毒(ASFV)MGF505-5R蛋白及其特异性多克隆抗体,分别将His-SUMO、His-TrxA、His-GST、His-MBP和His-NusA基因片段与MGF505-5R基因连接,构建原核表达载体pET28a-SUMO-MGF505-5R、pET28a-TrxA-MGF505-5R、pET28a-GST-MGF505-5R、pET28a-MBP-MGF505-5R和pET28a-NusA-MGF505-5R,并分别转化至大肠杆菌BL21(DE3)感受态细胞中进行诱导表达;通过对诱导剂浓度、诱导温度和诱导时间进行优化,研究最佳表达条件;采用麦芽糖亲和层析和Ni2+亲和层析对表达的重组蛋白进行纯化,并将纯化后的重组蛋白免疫BALB/c小鼠制备多克隆抗体;应用Western blot方法对制备的多克隆抗体进行鉴定。结果显示,MBP-MGF505-5R重组蛋白能够在大肠杆菌中可溶性表达,在20℃条件下,IPTG浓度为0.5 mmol/L、诱导12 h时,重组蛋白可溶性表达量最高;纯化后可获得纯度达90%以上的MGF505-5R蛋白。免疫小鼠后制备的多克隆抗体能够与MGF505-5R蛋白发生特异性反应。综上,利用大肠杆菌表达系统实现了ASFV MGF505-5R蛋白的可溶性表达,且纯化后重组蛋白纯度较高,具有生物学活性。

African swine fever virus MGF505-3R inhibits cGAS-STING-mediated IFN-βpathway activation by degrading TBK1

African swine fever virus(ASFV)is an important pathogen causing acute infectious disease in domestic pigs and wild boars that seriously endangers the global swine industry.As ASFV is structurally complex and encodes a large number of functional proteins,no effective vaccine has been developed to date.Thus,dissecting the mechanisms of immune escape induced by ASFV proteins is crucial.A previous study showed that the ASFV-encoded protein is an important factor in host immunity.In this study,we identified a negative regulator,MGF505-3R,that significantly downregulated cGAS/STING-and poly(dG:dC)-mediated IFN-βand interferon stimulation response element(ISRE)reporter activity and suppressed IFNB1 and IFIT2 mRNA levels.In addition,TBK1,IRF3 and IκBαphosphorylation levels were also inhibited.Mechanistically,MGF505-3R interacted with cGAS/TBK1/IRF3 and targeted TBK1 for degradation,thereby disrupting the cGAS-STING-mediated IFN-βsignaling pathway,which appears to be highly correlated with autophagy.Knockdown MGF505-3R expression enhanced IFN-βand IL-1βproduction.Taken together,our study revealed a negative regulatory mechanism involving the MGF505-3R-cGAS-STING axis and provided insights into an evasion strategy employed by ASFV that involves autophagy and innate signaling pathways.

Genetic variation of TGF-BR2 as a protective genotype for the development of colorectal cancer in men

BACKGROUND The role of transforming growth factor beta(TGF-β)signaling,including both the cytokine and their receptors,in the etiology of colorectal cancer(CRC)has been of particular interest lately.AIM To investigate the association between promoter polymorphism in TGF-β receptor 2 TGF-BR2G^([-875])A with a CRC risk in a cohort of Bulgarian patients using a casecontrol gene association study approach,as well as the protein levels of TGF-β1 in the peripheral blood.METHODS A cohort of 184 CRC patients and 307 sex and age-matched healthy subjects were recruited in the study.A genotyping of the TGF-BR2G^([-875])A(rs3087465)polymorphism was performed by primer-introduced restriction analysespolymerase chain reaction approaches.RESULTS The frequency of TGF-BR2G^([-875])A genotype was decreased in male patients with CRC than in healthy men(31.3%vs 44.8%;P=0.058).Among males,the TGF-BR2G[-509]G genotype was related to a significantly increased risk of CRC development(OR=1.820,95%CI:0.985-3.362,P=0.055)than the GA+AA genotype.Also,TGF-BR2^([-875])*A-allele itself was rarer in men with CRC than healthy men(19.1%vs 26.9%,P=0.086)and was associated with a protective effect(OR=0.644;95%CI:0.389-1.066;P=0.086).Regarding the genotypes,we found that TGF-β1 serum levels were higher in GG genotype in healthy persons above 50 years than the CRC patients[36.3 ng/mL interquartile range(IQR)19.9-56.5 vs 22.4 ng/mL IQR 14.8-29.7,P=0.014].We found significant differences between higher levels of TGF-β1 serum levels in healthy controls above 50 years(GG genotype)and CRC patients(GG genotype)at the early stage(36.3 ng/mL IQR 19.9-56.5 vs 22.8 ng/mL IQR 14.6-28.6,P=0.037)and advanced CRC(36.3 ng/mL IQR 19.9-56.5 vs 21.6 ng/mL IQR 15.9-33.9,P=0.039).CONCLUSION In summary,our results demonstrated that TGF-BR2 AG and AA genotypes were associated with a reduced risk of CRC,as well as circulating levels of TGF-βcould prevent CRC development in a gender-specific manner.Notably,male carriers of TGF-BR2-875A allele genotypes had a lower risk of CRC development and progression,suggesting that TGF-BR2-875A/G polymorphism significantly affects the protective biological factors that also impact the risk of colon and rectal carcinogenesis.

Induction of anti-hepatoma immunity by recombinant retrovirus expressing B7-1 /B7-2 costimulatory molecules

Objective: To construct recombinant R7-l/B7-2 retrovirus vectors and observe the effects of B7-l/R7-2 gene expression on in ho and in for immune response against against murine hepatoma. Methods: The recombinant retrovirus vectors expressing B7-1/B7-2 were constructed by gene cloning technology to produce retrovirus-infected PE501 and PA317 cell lines and murine hepatoma Hepal-6. The expression of R7-l/B7-2 was detected by fluorescence activated cell soning analysis (FACS). B7-l/B7-2 positive Hepal-6 Cell lines were used in inducing anti-hepatoma immunity in ho and in the. Results: In contrast to the excessive growth of parental Hemal-6 tumor, the growth of B7-l/B7-2-positive Hepal-6 inoculated into syngenic mice regressed. B7-1/R7-2-positive or cytokine-treated Hepal-6 alone could only induce mild cytototicity; in contrast, B7-1/B7-2-positive Hemal-6 treated with cytokine-stimulated spleen cells and activated the cytotoxicity effectively. Immunity in mice with R7-1/B7-2-positive tumor cells or cytokine-beated Hepal-6 only provided partial protection against parental Hepa1-6 tumor, whereas pretreatment of the transfected tumor cells with IFN-r and TNF-a induced complete immunity protection in vivo. Mice receiving inoculation of cytokine-treated B7-l/R7-2-positive Hemal-6 cells presented regression of the establoshed pental tUmor and survived for more than l00 d, while those untreated mice died within 40 d. Conclu sions: B7-l/R7-2 expression is necessary but not sufficient in inducing anti-hepatoma immune response, whereas it is efficient when combined with the beatment of IFN-γ and TNF-a.

Identification of African swine fever virus MGF505-2R as a potent inhibitor of innate immunity in vitro

African swine fever(ASF)is etiologically an acute,highly contagious and hemorrhagic disease caused by African swine fever virus(ASFV).Due to its genetic variation and phenotypic diversity,until now,no efficient commercial vaccines or therapeutic options are available.The ASFV genome contains a conserved middle region and two flexible ends that code for five multigene families(MGFs),while the biological functions of the MGFs are not fully characterized.Here,ASFV MGF505-2R-deficient mutant ASFV-Δ2R was constructed based on a highly virulent genotype II field isolate ASFV CN/GS/2018 currently circulating in China.Transcriptomic profiling demonstrated that ASFV-Δ2R was capable of inducing a larger number of differentially expressed genes(DEGs)compared with ASFV CN/GS/2018.Hierarchical clustering of up-regulated DEGs revealed that ASFV-Δ2R induced the most dramatic expression of interferon-related genes and inflammatory and innate immune genes,as further validated by RT-qPCR.The GO and KEGG pathway analysis identified significantly enriched pathways involved in pathogen recognition and innate antiviral immunity.Conversely,pharmacological activation of those antiviral immune responses by exogenous cytokines,including type I/II IFNs,TNF-αand IL-1β,exerted combinatory effects and synergized in antiviral capacity against ASFV replication.Collectively,MGF505-2R is a newly identified inhibitor of innate immunity potentially implicated in immune evasion.

非洲猪瘟病毒防污染双重荧光定量PCR检测方法的建立

荧光定量聚合酶链反应(qPCR)技术在非洲猪瘟病毒(ASFV)的检测中发挥重要作用,然而该方法也存在一些局限性。一方面由于检测环境中易产生气溶胶污染,经常导致qPCR检测产生假阳性结果。另一方面,临床上已发现MGF基因缺失的变异株,现有的检测方法将无法满足野毒株与变异株鉴别检测的需求。为了解决qPCR易产生假阳性,ASFV核酸检测靶标基因过分单一的问题,建立了ASFV的防污染双重qPCR检测方法。该检测方法根据C962R基因和MGF-505-2R基因的保守序列设计引物和探针,并在反应体系中加入UNG酶达到防污染的目的,建立了ASFV的防污染双重qPCR检测方法。结果表明,利用该方法对C962R基因和MGF-505-2R的基因标准品进行检测,该检测方法具有较好的鉴别诊断效果。该检测方法的标准曲线具有良好的线性关系,相关系数为0.99;敏感性高,最低检测限为10 copies/μL的ASFV基因标准品;特异性强,与猪丹毒丝菌(Erysipelothrix rhusiopathiae)、猪伪狂犬病病毒(PRV)、副猪嗜血杆菌(Hoemophilus parasuis)、猪链球菌(Streptococcus suis)、猪传染性胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)及猪圆环病毒2型(Porcine circovirus 2,PCV2)均无交叉反应;重复性较好,变异系数小于1.75%,使用该检测方法对194份临床组织样品检测,与商品化非洲猪瘟病毒抗原检测试剂盒比较,检测结果均为阴性,符合率为100%。该方法预期可用于ASFV野毒株与MGF-505-2R基因缺失株的鉴别诊断,为深入开展分子流行病学调查提供技术储备。

FoxJ1 inhibits African swine fever virus replication and viral S273R protein decreases the expression of FoxJ1 to impair its antiviral effect

African swine fever(ASF)is a highly pathogenic swine infectious disease that affects domestic pigs and wild boar,which is caused by the African swine fever virus(ASFV).ASF has caused huge economic losses to the pig industry and seriously threatens global food security and livestock health.To date,there is no safe and effective commercial vaccine against ASF.Unveiling the underlying mechanisms of ASFV-host interplay is critical for developing effective vaccines and drugs against ASFV.In the present study,RNA-sequencing,RT-qPCR and Western blotting analysis revealed that the transcriptional and protein levels of the host factor FoxJ1 were significantly down-regulated in primary porcine alveolar macrophages(PAMs)infected by ASFV.RT-qPCR analysis showed that overexpression of FoxJ1 upregulated the transcription of type I interferon and interferon stimulating genes(ISGs)induced by poly(dA:dT).FoxJ1 revealed a function to positively regulate innate immune response,therefore,suppressing the replication of ASFV.In addition,Western blotting analysis indicated that FoxJ1 degraded ASFV MGF505-2R and E165R proteins through autophagy pathway.Meanwhile,RT-qPCR and Western blotting analysis showed that ASFV S273R inhibited the expression of FoxJ1.Altogether,we determined that FoxJ1 plays an antiviral role against ASFV replication,and ASFV protein impairs FoxJ1-mediated antiviral effect by degradation of FoxJ1.Our findings provide new insights into the antiviral function of FoxJ1,which might help design antiviral drugs or vaccines against ASFV infection.

非洲猪瘟病毒野毒株与基因缺失株三重荧光定量PCR鉴别检测方法的建立及应用

针对非洲猪瘟病毒B646L、MGF505-2R和CD2v基因分别设计了引物和探针,经过条件优化,建立了基于TaqMan探针技术的三重荧光定量PCR检测方法。结果显示,本研究建立的三重荧光定量PCR检测方法与猪场常见病毒的核酸不发生交叉反应,具有良好的特异性;敏感性分析显示,针对B646L、MGF505-2R和CD2v基因的最低检测下限分别为6.5,8.0和14.0 copies/μL;并且该方法在10~10copies/μL模板范围内具有良好的线性关系,组间变异系数为0.05%~2.68%,组内变异系数为0.10%~1.17%,稳定性良好。进一步针对临床核酸样品的检测显示,本方法和OIE推荐的检测方法具有良好的一致性。本研究成功建立了非洲猪瘟病毒野毒株和基因缺失株的荧光定量PCR鉴别检测方法,为临床非洲猪瘟病毒的监测诊断提供了良好的技术支撑。