【作者】 王彦彬；

【导师】 崔保安；

【作者基本信息】 河南农业大学 ， 预防兽医， 2008， 博士

【摘要】 一、为获得猪干扰素α(Porcine interferon alpha,PoINF-α)和干扰素γ(Porcine interferon gamma,PoINF-γ)基因,本研究参照Genebank已发表的猪干扰素α和干扰素γ基因序列,分别设计一对引物,应用反转录多聚酶链式反应,从有丝分裂原刀豆素(ConA)诱导的猪脾淋巴细胞总RNA中扩增出猪干扰素α和干扰素γ基因,将扩增片段通过T-A策略连接到克隆质粒pGEM-T载体上,经PCR鉴定、酶切鉴定和序列测定,证明克隆片段为猪干扰素α和干扰素γ基因。与网上公布的猪干扰素α和干扰素γ基因序列进行比较,其序列同源性分别99.8%和100%。从而为进一步应用该基因在杆状病毒/昆虫细胞表达系统中进行表达奠定了基础。二、杆状病毒/昆虫细胞是被广泛用来表达外源基因的表达系统,但许多表达的外源蛋白存在于细胞内,不能分泌到细胞上清。Tessier研究发现蜂素信号肽能够介导外源基因进行分泌表达,但又有该信号肽无效的报道,因此本课题的目的是研究蜂素信号肽对猪干扰素α在昆虫细胞中表达是否产生影响。根据蜂素信号肽序列,设计二条部分重叠的引物,通过PCR扩增出蜂素信号肽基因,然后与编码成熟猪干扰素α的基因片段通过PCR进行连接获得嵌合体基因片段,同时扩增出即不包含蜂素信号肽又不包含原有信号肽的仅编码成熟猪干扰素α的基因片段,将二个片段分别插入pFastBacTMDual载体,置于pH启动子控制下,酶切和测序验证正确后,转化到DH10Bac宿主菌,在三个抗性和IPTG、X-gal的共同作用下,通过定点转位获得穿梭载体bacmid。重组bacmid经电泳和PCR鉴定,然后转染昆虫细胞,获得重组杆状病毒。间接免疫荧光和Western blot鉴定重组蛋白的表达,结果表明,带有蜂素信号肽的猪干扰素α在昆虫细胞中实现分泌表达,而不带蜂素信号肽的猪干扰素α重组蛋白主要分布在昆虫细胞内。从而证明蜂素信号肽能够介导猪干扰素α在昆虫细胞中的分泌表达。通过猪水泡性口炎病毒(VSV)在猪肾细胞(PK-15)致病变抑制作用检测其抗病毒活性,表明带有蜂素信号的重组杆状病毒感染昆虫细胞获得的重组蛋白对VSV表现出较高的抑制活性,上清的抗病毒效价达到1.07×106 U/mL。昆虫细胞裂解液的抗病毒效价为3.15×105 U/mL。上清中抗病毒活性为细胞中的抗病毒活性近3.4倍。不带蜂素信号肽的重组杆状病毒感染昆虫细胞获得的重组蛋白,细胞中的抗病毒活性为3.82×105 U/mL,上清中的抗病毒活性为9.67×104 U/mL。细胞中的抗病毒活性高于上清中的抗病毒活性。对比带蜂素信号肽和不带蜂素信号肽对猪干扰素α在昆虫细胞中的表达,蜂素信号肽能够介导猪干扰素α在昆虫细胞中达到分泌型表达,并且对表达具有增强作用,增强能力达到近3倍。三、为获得重组猪γ干扰素,本研究应用Bac-to-Bac杆状病毒/昆虫细胞表达系统,将编码成熟猪干扰素γ基因插入供体质粒pFastBacTMDual多克隆位点,置于pH启动子控制下,为实现分泌型表达,昆虫细胞可识别的蜂素信号肽取代猪α干扰素原有信号肽构建嵌合体基因,并在C端融合6个组氨酸标签以便于纯化。将构建质粒转化DH10Bac感受态细胞,在三个抗性作用下,通过蓝白斑筛选,获得重组Bacmid穿梭质粒,转染对数生长期的Sf9昆虫细胞获得重组杆状病毒。猪γ干扰素重组蛋白通过间接免疫荧光、Western-blot证明在重组杆状病毒感染的昆虫细胞中获得分泌表达。通过在猪肾细胞(PK-15)上抑制水泡性口炎病毒(VSV)致病变作用检测重组蛋白的抗病毒活性,结果表明:昆虫细胞上清的抗病毒效价达到6.67×105 U/mL。重组猪γ干扰素的获得为进一步作为免疫佐剂对各种疫苗的增强作用,以及用作治疗剂在临床上治疗病毒性疫病等方面的研究奠定了基础。四、猪干扰素包含有α、β和γ三种,其中α、β属于Ⅰ型,具有较强的抗病毒作用,是由受病毒感染的多种体细胞产生,γ属于Ⅱ型干扰素,除具有较强的抗病毒作用外,还具有较强的免疫调节作用,主要是由被激活的T细胞和NK细胞产生。Ⅰ型干扰素和Ⅱ型干扰素联合使用,具有协同作用,比单独使用其中一种具有更强的抗病毒效果。为获得Ⅰ型和Ⅱ型复合型干扰素重组蛋白,本研究利用Bac-to-Bac杆状病毒表达系统将PoINF-α和PoINF-γ基因克隆到杆状病毒转移载体pFastBacTMDual中,分别置于pH和p10启动子控制下,为达到分泌型表达,二个基因的原有信号肽通过PCR技术替换为昆虫细胞可以识别并剪切的蜂素信号肽。获得重组转移载体后,再将其转化进含穿梭载体Bacmid的感受态细胞DH10Bac中,经蓝白菌落筛选获得含有二个外源基因的重组穿梭载体rBacmid,经脂质体介导转染昆虫细胞Sf9,获得包含二个外源基因的重组杆状病毒。SDS-PAGE和Western-Blotting分析可见大小约为19kDa-21kDa的特异性条带及其它不同糖基化异构体。间接免疫荧光发现荧光信号主要分布在细胞膜上,证明是分泌型表达。抗病毒活性检测发现重组蛋白具有极强的抑制VSV病毒在PK-15细胞上增殖活性,上清抗病毒达到3.24×107 U/mL,细胞中抗病毒活性为5.86×106 U/mL。五、猪繁殖与呼吸综合征(Porcine Reproductive and Respiratory Syndrome,PRRS)是由猪繁殖与呼吸综合征病毒引起的主要导致怀孕母猪后期流产、早产、死胎和木乃伊胎产弱仔增多,仔猪出现咳嗽、呼吸困难、呼吸急促等呼吸道症状和断奶前死亡率增高的一类疾病,在世界范围内广泛存在,给养猪业创成巨大的经济损失。许多报道证明干扰素对猪蓝耳病病毒具有抑制作用。本研究制备的复合型干扰素在Marc-145细胞上对猪蓝耳病病毒是否具有抑制作用。通过致病变抑制作用进行检测,结果表明复合型干扰素具有较强的抗病毒作用,干扰素粗制品经210稀释后,在Marc-145细胞上能够抑制猪蓝耳病病毒100TCID50的致细胞病变作用。更多还原

【Abstract】 1: Clone of Porcine Interferon-αand Interferon-γGenesTwo pairs of RT-PCR primers were designed and synthesized based on the published gene sequence of porcine interferon-α(PoIFN-α)and interferon-γ(PoIFN-γ).The two about 500bp target DNA sequences were amplified by reverse transcription polymerase chain reaction depending on the template of total RNA which isolated from ConA-stimulated porcine spleen cell,and cloned into pGEM-T vector.The two gene fragment were identified to be interferon-αand interferon-γwith the methods of restriction enzyme digestion,polymerase chain reaction,and sequencing.Comparing with the sequences of porcine that deposited in NCBI.There are completely consensus with these. That will allow us to explore this cytokine as a poteintial therapeutic agent for porcine diseases using a baculovirus/insect cell expression system.2: Effects of the honeybee melittin signal peptide on expression of porcine interferon-αin insect cellsThe baculovirus-insect cell system is a valuable tool for the expression of heterologous proteins. Due to limitations in the intracellular processing environment, however, heterologous secreted and membrane proteins are often insoluble.Many attempts to modify the insect cell secretory pathway by overexpressing processing factors have demonstrated the potential to overcome these limitations.Report by Tessier and his co-worker showing that secretion of a plant protein in the baculovirus system was enhanced when its signal peptide was replaced with an insect-derived signal peptede: the honeybee melittin signal peptide(HBM).However, There are studies showed that insect-derived signal peptides and/or prosequences cannot always enhance the expression and/or secretion of foreign secretory pathway proteins in the baculovirus system. The aim of this study was to investigate the effects of the honeybee melittin signal peptide on baculovirus-mediated expression and secretion of porcine interferon-α.The HBM gene was amplified by PCR using a pair of primers with partial overlapping sequence.The synthetic chimaera was obtained by ligation the sequence coding mature porcine interferon-αwith HBM by PCR.The other fragment without HBM was amplificated also.Each gene was cloned into the baculovirus transfer vector pFastBacTMDual,under the control of pH promoter. Constructs were transformed into DH10BacTM E.coli,the site-specific transposition occurs between the mini-Tn7 element on the pFastBacTMDual vector and the mini-attTn7 target site on the bacmid to generate a recombinant bacmid ,then introduced to Spodoptera frugiperda.Immunofluorescence assay and western blot analysis confirmed the secretory expression of recombinant baculovirus with HBM.Overxpression of the porcine interferon-αform has also been achieved using recombinant baculovirus without HBM but mostly in an aggregated form with no secretion. Using the honeybee melittin signal peptides in baculovirus expression vectors is enable to aid in increasing expression and yield of heterologous secreted proteins in insect cells.3: Secretory Expression of Porcine Interferon-γusing a baculovirus system and its antiviral activity detectionCytokines, such as porcine interferon-gamma(PoIFN-γ) have been shown to have antiviral and adjuvant activity in livestock and have the potential to be used as alternatives to antibiotics. This part aimed to the expression of PoIFN-γin a baculovirus system to generate a fully functional recombinant protein. Porcine interferon-γcDNA was cloned from mitogen stimulated spleen lymphocytic total RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The sequences encoding for a mature 145 amino acid protein in frame with a C-terminal 6×Histidine tag,was cloned and expressed into baculovirus transfer vector pFastBacTMDual,under the control of pH promoter.The authentic signal sequences of porcine interferon-γwere substituted with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell.The recombinant proteins were successfully detected in expressing cells by immunofluorescence assay and in the culture medium by western blot analysis.The recombinant PoIFN-γis verified to be of high cytokine activity by inhibiting the cytopathic effect of vesicular stomatitis virus in PK-15 cells, which is about 6.67 x 10(5) U/mL in supernatant.4: Co-expression of porcine interferon-α&γusing a baculovirus system and its antivirus activity detectionPorcine interferon(interferon,IFN) comprises IFN-α, IFN-βand IFN-γ.Type I IFNs (IFN-αand IFN-β) and type II IFN (IFN-γ) are important components of the host immune response to viral infections. IFN-αand IFN-βare produced by most cells as a direct response to viral infection, while IFN-γis synthesized almost exclusively by activated NK cells and activated T-cells in response to virus-infected cells.Like type I IFNs,IFN-γhas antiviral activity,but in addition it also posses immunomodulatory activity. Combination of type I and type II interferons act synergistically such a mixture of the two has much greater activity than that expected from the separate contribution of each type. Baculovirus/insect cell system was used for such mixture recombinant proteins production.Porcine interferon-αgene and interferon-γgene were cloned into pFastBacTMDual vector that has two promoters and cloning sites,allowing simultaneous expression of these two genes.The two proteins in frame with a C-terminal hexahistidine(His6) tag to facilitate purification of the secreted protein on nickel-containing resins. The authentic signal sequences of the two genes were substituted with the honeybee melittin signal sequence, allowing efficient entrance into the secretory pathway of the insect cell.Both genes were cloned into the baculovirus transfer vector pFastBacTMDual,under control of pH promoter and p10 promoter,respectively.A recombinant baculovirus,Bac-PoIFN-α&γ, involving two heterogenous genes, was generated by transfecting pBac-PoIFN-α&γto bacmid inside DH10Bac Escherichia coli by site-specific transposition,followed by transfection into the sf9 cells.The expressed recombinant glycoproteins were successfully detected in expressing cells by SDS-PAGE and by western blot analysis. Secretory expression of the recombinant protein was verified by immunofluorescence assay that indicating immunofluorescence signal mainly locate at cytomembrane.The bioactivity was confirmed to be of high cytokine activity by inhibiting the cytopathic effect of vesicular stomatitis virus in PK-15 cells, which is about 3.24×107 U/mL in supernatant and 5.86×106 U/mL in insect cells, respectively.5: Inhibition of porcine reproductive and respiratory syndrome virus in Marc-145 cells by compound of recombinant porcine interferon.Porcine reproductive and respiratory syndrome (PRRS) is caused by an enveloped positive-stranded RNA virus placed in the family Arteriviridae,Infection of adult pigs generally produces a nonfatal disease characterized by flu-like symptoms, including mild interstitial pneumonia, elevated temperature, and inappetance. In sharp contrast, the infection of pregnant gilts and sows results in abortion and the birth of dead and weak-born piglets.Surviving piglets exhibit the severest form of respiratory distress with mortality often approaching 100% within 3 weeks after farrowing. Surviving pigs continue to suffer the negative effect of PRRSV by exhibiting increased susceptibility to secondary infection. Pigs surviving acute PRRS support a long-term asymptomatic infection, which has contributed to the worldwide spread of the disease. The persistent infections result large economics loss in pig raising.Rowland reported that pretreatment of MARC-145 cells with IFN-γinhibited wild-type (SDSU-23983 P6) and culture-adapted (SDSU-23983 P136) PRRS viruses in a dose-dependent manner and at relatively low concentrations. The effect of IFN-γon virus replication included reductions in the number of infected cells, virus yield, and RNA content in single cells.The purpose of this part is to investigate the inhibited effect of recombinant compound IFN on PRRSv in Marc-145 cells with the cytopathic effect.The result show that the dilution of 210 of crude recombinant supernant can protect the Marc-145 from the cytopathic effect cause by 100TCID50 of Porcine reproductive and respiratory syndrome virus.更多还原