猪CD151转基因PK-15细胞系构建及其对猪繁殖与呼吸综合征病毒的易感性

[目的]建立猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)易感的猪CD151转基因PK-15细胞系,研究CD151分子在PRRSV感染猪源细胞中的作用.[方法]用RT-PCR从猪肺泡巨噬细胞中扩增CD151全长cDNA,测序正确后克隆人真核表达载体pcDNA3;用重组载体pcDNA-CD151转染PK-15细胞,经G418抗性筛选获得转基因细胞系PK15-CD151,用RT-PCR和免疫荧光试验检测CD151表达;用VR-2332株PRRSV分别感染PK-15细胞、PK15-CD151细胞、MARC-145细胞和3D4-CD163细胞,定期观察细胞病变,用RT-PCR和免疫荧光试验检测病毒RNA基因组和病毒抗原,用半数组织细胞感染剂量测定病毒滴度.[结果]从猪巨噬细胞中克隆得序列正确的猪CD151 cDNA;从重组载体转染的PK-15细胞培养中筛选得G418抗性细胞克隆,并能正确表达猪CD151分子;在PRRSV感染后,PK15-CD151细胞虽然不表现明显的细胞病变,但能检测到病毒RNA基因组和病毒抗原,并能产生较高滴度的感染性病毒;该细胞系已在体外传30代以上,第10、20、30代细胞的PRRSV滴度无明显变化.[结论]猪CD151基因转染能使非易感PK-15细胞获得对PRRSV的易感性,提示猪CD151参与PRRSV感染猪源细胞.     

猪源Viperin蛋白抑制猪繁殖与呼吸综合征病毒感染MARC-145细胞

为了研究干扰素刺激基因Viperin对猪繁殖与呼吸综合征病毒的抗病毒作用,本研究构建了猪源Viperin基因(sViperin)pCI-sVIP表达载体,并在MARC-145细胞上观察了其对PRRSV复制的抑制作用。结果为:过表达sViperin蛋白可以抑制PRRSV在MARC-145细胞上的复制,且具有剂量依赖作用。sViperin蛋白抑制了病毒的入胞,但其对病毒的装配和释放没有影响作用。共聚焦实验证明sViperin蛋白主要定位于内质网等细胞器。sViperin蛋白和PRRSV GP5、N蛋白在细胞内存在共定位现象。CO-IP实验证明sViperin蛋白可以和PRRSV N蛋白存在相互作用。该研究为该病毒新型抗病毒药物研究奠定了重要基础。     

犬细小病毒NS1 非结构蛋白可诱导细胞凋亡

【目的】研究犬细小病毒(Canine parvovirus,CPV)非结构蛋白NS1在CPV引起宿主细胞凋亡中的作用,初步探讨CPV引起细胞凋亡的机制。【方法】首先采用PCR方法从犬细小病毒基因组中扩增NS1编码基因,然后利用pcDNA3.1A质粒构建NS1真核表达载体pcDNA-NS1,并通过HEK293FT细胞瞬时表达NS1重组蛋白,用Western-blot检测以确定重组NS1蛋白能否在真核细胞中表达。然后用CPV感染和用pcDNA-NS1表达载体转染F81宿主细胞,通过AnnexinV/PI双染法检测磷脂酰丝氨酸外翻和通过化学发光法检测caspase-3/7活性,分析感染CPV或转染NS1基因对F81宿主细胞凋亡的影响。【结果】结果表明,本实验扩增的NS1基因序列与GenBank的序列一致,构建的表达载体结构正确,并能够介导NS1基因在真核细胞中表达。感染CPV和转染NS1基因均能诱导F81细胞膜磷脂酰丝氨酸外翻和明显提高细胞内caspase-3/7的活性,表明CPV和NS1蛋白均能引起细胞的凋亡。【结论】CPV诱导宿主细胞凋亡与其编码的NS1非结构蛋白有关。     

结核分枝杆菌38kD蛋白真核表达载体的构建及其在HEK293T细胞中的分泌性表达

目的:构建含结核分枝杆菌38kD蛋白基因的真核表达载体并转染HEK293T细胞,高效表达分泌性38kD蛋白。方法:设计合成的结核分枝杆菌38kD基因被克隆到T载体,然后亚克隆到真核表达载体pcDNA3.0,经酶切鉴定正确后,PEI转染法导入293T细胞,换不含血清的DMEM培养基培养3 d后收集细胞及上清,采用Western印迹检测38kD蛋白的表达。结果:酶切结果显示,获得正确的含38kD基因的重组表达载体;Western印迹结果显示表达载体导入293T细胞中后能在细胞及上清中检测到38kD蛋白表达。结论:构建了含重组结核分枝杆菌38kD蛋白基因的真核表达载体pcDNA3.0-38kD,该载体可在哺乳动物细胞HEK293T中高效分泌性表达38kD蛋白,为结核病诊断试剂盒研发奠定了基础。     

基于逆转录病毒载体的外源基因表达系统的评价和应用

逆转录病毒表达系统是基因治疗研究和RNA干扰技术广泛采用的外源基因表达系统。文中以增强型绿色荧光蛋白 (EGFP) 基因的表达水平和稳定性为指标,比较逆转录病毒表达载体pQCXIN和pcDNA3.1(+) 表达质粒介导的外源基因在HEK293细胞和CHO-K1细胞的表达效率。病毒感染HEK293细胞和CHO-K1细胞的相对荧光强度 (Relative fluorescence intensity,RFI) 均约为对应的质粒转染细胞的2倍。多轮反复感染逆转录病毒表达载体能有效提高HEK293细胞表达EGFP的效率。HEK293细胞经4轮病毒感染后的RFI值较1次病毒感染HEK293细胞的RFI值约提高2倍。此外,逆转录病毒表达载体介导的外源基因表达的稳定性优于质粒转染的外源基因表达。采用携带人重组活性蛋白C (Recombinant human activated protein C,rhAPC) 基因的pQCXIN和HEK293细胞进一步验证了逆转录病毒载体介导的外源基因表达效率,构建了rhAPC表达水平为10~15 mg/(106 cells·d) 的HEK293细胞系。研究结果表明,逆转录病毒表达系统是有应用价值的介导外源基因在哺乳动物细胞高效表达的技术途径。     

犬细小病毒VP2蛋白在真核细胞中的分泌表达及特性

摘要:【目的】利用真核细胞分泌表达犬细小病毒VP2蛋白和研究其特性。【方法】为构建犬细小病毒（Canine parvovirus, CPV）VP2基因的真核分泌型表达载体,首先通过酶切从含有人CD5信号肽序列的质粒中将CD5信号肽基因片段切出,将其连接到真核表达载体pcDNA3.1A的多克隆位点上,构建成pcDNA3.1-CD5sp质粒。然后再通过PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2基因,并将其插入到pcDNA3.1- CD5sp载体中CD5信号肽的下游,构建成VP2基因的真核分泌型表达载体pcDNA-CD5sp-VP2。经磷酸钙介导转染293T细胞,使其在真核细胞中进行分泌表达,并通过ELISA检测表达的VP2蛋白与犬转铁蛋白受体（TfR）结合的活性。【结果】序列分析结果表明,本实验构建的犬细小病毒VP2基因真核分泌型表达载体结构正确,将该表达载体转染的293T细胞,在培养基中通过Western-blot检测到有VP2重组蛋白的存在。经ELISA检测表明表达的重组VP2蛋白具有与犬转铁蛋白受体结合的活性。【结论】 利用人的CD5信号肽实现了犬细小病毒VP2蛋白在真核细胞中的分泌表达,表达的VP2蛋白具有与犬转铁蛋白受体结合的活性。     

人Rab12基因真核表达载体的构建及在HEK293细胞中的表达

Rab蛋白参与细胞的囊泡运输过程。自噬体-溶酶体的融合需要活化的Rab12蛋白参与。本研究从HEK293细胞获取总RNA,采用RT-PCR方法扩增出Rab12基因的ORF全序列,将其克隆到含有增强型绿色荧光蛋白(EGFP)基因的真核表达载体pEGFP-N1上,成功构建重组质粒pEGFP-Rab12,然后采用lipofectamine 2000将重组质粒转染至HEK293细胞中并获得高表达。     

猪骨髓基质抗原2基因的克隆、表达及生物学活性研究

研究目的是克隆猪骨髓基质抗原2(Bone marrow stromal antigen-2,BST-2)基因并进行真核表达,获得具有抗病毒活性的重组BST-2蛋白。研究通过RT-PCR从猪瘟弱毒疫苗和聚肌胞(Poly I:C)刺激的猪肾细胞(PK-15)中扩增出猪BST-2cDNA,克隆至真核表达载体pcDNA3.1/V5-His,构建重组表达质粒pcDNA-BST-2,转染HEK293T细胞,纯化BST-2蛋白并经Bradford法定量,Western blot检测,研究BST-2抗病毒生物学活性。酶切鉴定和核酸序列测定证实pcDNA-BST-2真核表达质粒构建成功,转染HEK293T细胞后,经间接免疫荧光能够检测到绿色荧光。生物学活性测定重组BST-2蛋白具有一定的抗水泡性口炎病毒(Vesicular stomatitis virus,VSV)、H9禽流感病毒(Avian influenza virus,AIV)及猪繁殖与呼吸综合症病毒(Porcine reproductive and respiratory syn-drome virus,PRRSV)的活性。结果表明,重组BST-2具有一定抗病毒生物功能,为进一步研究重组BST-2蛋白的活性以及BST-2抗病毒药物研究奠定了基础。     

USP22基因克隆及其融合蛋白真核表达载体的构建

目的克隆人类泛素水解酶22（ubiquitin—specific processing enzyme22,USP22）基因,构建与绿色荧光蛋白融合表达的真核载体。方法利用RT—PCR技术以HeLa细胞总RNA为模板分别扩增USP22基因cDNA序列两片段,依次插入真核表达载体pEGFP—N1;重组质粒转染HEK293T细胞,观察融合蛋白表达及绿色荧光的分布。结果测序结果显示USP22序列与GenBank收录数据一致,酶切显示重组质粒pEG—FP—USP22构建无误,质粒转染后有80％左右HEK293T细胞表达绿色荧光,且在胞质与胞核中广泛分布。结论成功克隆USP22基因并构建与GFP融合表达的真核载体,为进一步深入研究USP22基因的生物学功能奠定了基础。     

SCG10基因重组质粒的构建及蛋白表达和定位

目的构建SCG10真核表达载体并证实融合蛋白在细胞内表达及定位。方法以人胎脑cDNA文库为模板,PCR扩增SCG10全长编码基因,亚克隆至pEGFP-C1表达载体中。将构建的重组质粒测序并转染到人胚肾HEK293中,提取细胞蛋白进行Western blot检测。利用共聚焦激光扫描显微镜观察pEGFP-SCG10在HEK293细胞内定位。结果 SCG10全长基因序列克隆到了真核表达载体pEGFP-C1中,酶切鉴定片段大小540bp。Western blot检测到了融合蛋白表达,分子量约为48kD。pEGFP-SCG10在细胞内定位以细胞浆为主,在细胞核少量表达。结论成功构建了SCG10全长基因真核表达载体,pEGFP-SCG10蛋白主要定位于HEK293细胞浆内。     

3′非翻译区靶向人工微小RNA对PRRSV在猪肺巨噬细胞中复制的抑制作用（英文）北大核心CSCD

【目的】研究重组腺病毒(rAd)传送的3′非翻译区(UTR)靶向amiR3UTR对猪繁殖与呼吸综合征病毒(PRRSV)在猪肺巨噬细胞(PAM)中复制的抑制作用。【方法】用表达amiR3UTR或对照amiRcon的腺病毒载体转染AAV-293细胞,获得rAd-amiR3UTR-GFP和rAd-amiRcon-GFP,用定量RT-PCR检测amiR3UTR在rAd转导细胞中的表达,用定量RT-PCR、Western blotting和病毒滴定检测amiR3UTR对PRRSV复制的抑制作用。【结果】原代PAM及其细胞系3D4/163均能被rAd-amiR3UTR-GFP转导,但前者转导效率很低;rAd-amiR3UTR-GFP转导细胞能有效表达amiR3UTR,且表达具有剂量和时间依赖性;rAd表达的amiR3UTR能显著抑制不同毒株PRRSV在PAM细胞中的复制,且抑制作用具有剂量依赖性。【结论】amiR3UTR能抑制不同毒株PRRSV在PAM中的复制,其rAd有望作为抗PRRSV新策略进行深入研究。     

CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses

Direct functional screening of a cDNA expression library derived from primary porcine alveolar macrophages (PAM) revealed that CD163 is capable of conferring a porcine reproductive and respiratory syndrome virus (PRRSV)-permissive phenotype when introduced into nonpermissive cells. Transient-transfection experiments showed that full-length CD163 cDNAs from PAM, human U937 cells (histiocytic lymphoma), African green monkey kidney cells (MARC-145 and Vero), primary mouse peritoneal macrophages, and canine DH82 (histocytosis) cells encode functional virus receptors. In contrast, CD163 splice variants without the C-terminal transmembrane anchor domain do not provide PRRSV receptor function. We established several stable cell lines expressing CD163 cDNAs from pig, human, and monkey, using porcine kidney (PK 032495), feline kidney (NLFK), or baby hamster kidney (BHK-21) as the parental cell lines. These stable cell lines were susceptible to PRRSV infection and yielded high titers of progeny virus. Cell lines were phenotypically stable over 80 cell passages, and PRRSV could be serially passed at least 60 times, yielding in excess of 10(5) 50% tissue culture infective doses/ml.     

Actinobacillus pleuropneumoniae Possesses an Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV pre-infection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (<1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon γ. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools.     

The design and recombinant protein expression of a consensus porcine interferon: CoPoIFN-α

CoPoIFN-α is a recombinant non-naturally occurring porcine interferon-α (IFN-α). It was designed by scanning 17 porcine IFN-α nonallelic subtypes and assigning the most frequently occurring amino acid in each position. We used a porcine IFN-α (PoIFN-α) derived from domestic pig as a control. Both porcine IFN-α genes were introduced into yeast expression vector PpICZα-A and expressed in Pichia pastoris. The antiviral unit of these two IFN-αs were assayed in MDBK, PK-15 and MARC-145 cells against vesicular stomatitis virus (VSV), and their inhibitory abilities on pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) replication were also examined, respectively. We found the antiviral activity (units/mg) of CoPoIFN-α was 46.4, 63.6 and 53.5-fold higher than that of PoIFN-α for VSV inhibition in MDBK, PK-15 and MARC-145 cells, 4.8-fold higher for PRV inhibition in PK-15 cells, and 5-fold higher for PRRSV inhibition in MARC-145 cells. Our results also showed that the PRV and PRRSV-specific cytopathic effect (CPE) could be inhibited in the cells pretreated with CoPoIFN-α and PoIFN-α, and the virus titers in the cells pretreated with CoPoIFN-α were lower than those cells pretreated with PoIFN-α by 10-20-fold. The antiproliferative activity of CoPoIFN-α was significantly higher than that of PoIFN-α on a molar basis. The mRNA level of Mx1 and OAS1 genes in PK-15 cells induced by CoPoIFN-α were enhanced about 4.6-fold and 3.2-fold compared to that induced by PoIFN-α. Based on a homology model of CoPoIFN-α and IFNAR2, all of the different residues between native PoIFN-α and CoPoIFN-α were not involved in IFNAR1 binding site, and there is no direct interaction between these residues and IFNAR2, either. We speculate that the higher activity of CoPoIFN-α was likely due to the electrostatic potential introduced by residue Arg156 around the binding site or a structural perturbation caused by these different residues. This may enhance the overall binding affinity of CoPoIIFN-α and the receptors. Thus, CoPoIFN-α may have the potential to be used in therapy of porcine diseases.     

猪内源性反转录病毒囊膜基因env真核表达质粒的构建及鉴定

目的:构建猪内源性反转录病毒(PERV)囊膜基因env的真核表达质粒pHCMV-env并加以鉴定,为研究PERV的细胞嗜性和宿主范围奠定基础。方法:用RT-PCR方法扩增五指山猪来源PERV的env基因,将其插入pGEM-T easy载体中,构建重组质粒pGEM-T-env,酶切鉴定正确后,将pGEM-T-env与pHCMV-VSV-G表达质粒同时经EcoRⅠ酶切消化后连接,构建重组表达质粒pHCMV-env,并进行酶切、测序鉴定;将鉴定正确的质粒pHCMV-env转染HEK293T细胞,采用PCR、RT-PCR检测转染后env基因的整合和转录情况。结果:扩增得到五指山猪来源PERV的env基因,并构建了pHCMV-env真核表达质粒,转染HEK293T细胞系后,该细胞系中有目的基因的整合和转录。结论:构建了真核表达质粒pHCMV-env,并且在HEK293T细胞中能够整合并转录,为研究PERV的细胞嗜性和宿主范围奠定了基础。     

SOCS3基因重组腺病毒的构建及其在猪脂肪细胞中的表达

本研究旨在构建细胞因子信号转导抑制因子3(Suppressor of cytokine signaling 3,SOCS3)的重组腺病毒表达载体,获得有感染性的病毒颗粒。以pcDNA3-SOCS3质粒为模板扩增SOCS3基因,将其亚克隆至腺病毒穿梭载体pAdTrack-CMV,经测序验证后,重组的穿梭质粒用PmeI酶切线性化后转化到BJ5183感受态细菌中与其内的骨架载体pAdEasy-1进行同源重组,获得的重组质粒pAd-SOCS3,经PacI线性化后转染至HEK293细胞中进行包装和扩增,纯化后用TCID50法测定病毒滴度。以重组的病毒感染原代培养的猪脂肪细胞后,荧光显微镜下观察报告基因GFP的表达,RT-PCR和Western blotting检测细胞内SOCS3 mRNA和蛋白的表达。重组腺病毒载体pAd-SOCS3经酶切及PCR鉴定正确,病毒滴度为1.2×109PFU/mL;感染原代培养的猪脂肪细胞后,荧光显微镜观察可见报告基因GFP的表达;RT-PCR和Western blotting检测到细胞中SOCS3 mRNA和蛋白的表达显著提高。本研究成功构建了SOCS3基因的重组腺病毒,感染原代培养的猪脂肪细胞可稳定表达SOCS3蛋白,为深入研究SOCS3的功能奠定了基础。     

反转录病毒载体介导的猪APOBEC3F在猪源细胞PK15中的表达

目的:利用反转录病毒载体构建猪载脂蛋白B mRNA编辑酶催化多肽样蛋白(APOBEC)3F重组质粒,并实现其在猪肾细胞PK15中的表达。方法:用RT-PCR方法扩增五指山猪来源的外周血淋巴细胞APOBEC3F基因,将其定点插入反转录病毒载体pMSCV neo中,同时于插入位点两侧分别添加FLAG和GFP标签,构建重组质粒pMSCV-FLAG-A3F-GFP,并进行酶切、测序鉴定;将鉴定正确的重组质粒与pVSV-G、pGag-Pol共转染包装细胞HEK293T,分别于转染后48~72 h收集细胞的培养上清以获得假型病毒粒子;用该假型病毒感染猪源细胞PK15,通过PCR、Western印迹检测目的基因的整合及表达。结果:PCR扩增到1254 bp的猪APOBEC3F基因,重组质粒pMSCV-FLAG-A3F-GFP经酶切、测序,结果无误;3质粒共转染HEK293T细胞包装出的假型病毒感染PK15细胞后观察到GFP表达;从感染假型病毒的PK15细胞基因组中扩增到1254 bp的猪APOBEC3F基因,Western印迹检测到78.1×103的猪APOBEC3F蛋白的表达。结论:实现了反转录病毒载体介导的猪APOBEC3F在猪源细胞PK15中的整合与表达,为深入研究该分子对猪内源性反转录病毒(PERV)的抑制作用奠定了基础。     

Role of lipid rafts in porcine reproductive and respiratory syndrome virus infection in MARC-145 cells

Lipid rafts play an important role in the life cycle of many viruses. Cholesterol is a critical structural component of lipid rafts. Although the porcine reproductive and respiratory syndrome virus (PRRSV) has restricted cell tropism for cells of the monocyte/macrophage lineage, a non-macrophage cell MARC-145 was susceptible to PRRSV because of the expression of virus receptor CD163 on the cell surface, therefore MARC-145 cells is used as model cell for PRRSV studies. In order to determine if cholesterol is involved in PRRSV infection in MARC-145 cells, we used three pharmacological agents: methyl-β cyclodextrin (MβCD), mevinolin, and filipin complex to deplete cholesterol in MARC-145. Although these agents act by different mechanisms, they all significantly inhibited PRRSV infection. The inhibition could be prevented by addition of exogenous cholesterol. Cell membrane cholesterol depletion after virus infection had no effect on PRRSV production and cholesterol depletion pre-infection did not reduce the virus attachment, suggesting cholesterol is involved in virus entry. Further results showed that cholesterol depletion did not change expression levels of the PRRSV receptor CD163 in MARC-145, had no effect on clathrin-mediated endocytosis, but disturbed lipid-raft-dependent endocytosis. Collectively, these studies suggest that cholesterol is critical for PRRSV entry, which is likely to be mediated by a lipid-raft-dependent pathway.     

Intracellularly expressed nanobodies against non-structural protein 4 of porcine reproductive and respiratory syndrome virus inhibit virus replication

Objective

To isolate specific nanobodies to porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 4 (Nsp4) and investigate their potential antiviral activities.

Results

Three PRRSV Nsp4-specific nanobodies were isolated from a phage display library of the variable domains of camelid heavy chain-only antibodies. Nanobody genes were introduced into MARC-145 cells using lentivirus vectors to establish cell lines stably expressing nanobodies. These intracellularly expressed nanobodies were tested for interaction with PRRSV-encoded Nsp4 within PRRSV-infected MARC-145 cells. Nb41 and Nb43 intrabodies each potently inhibited PRRSV replication, protected MARC-145 cells from PRRSV-induced cytopathic effect and fully blocked PRRSV replication at an MOI of 0.001 or lower.

Conclusion

Intracellularly expressed Nb41 and Nb43 potently suppressed PRRSV replication in MARC-145 cells. Nanobodies hold great potential for development as novel antiviral treatments for PRRSV infection.