重组人肝刺激物在原核细胞中的表达与纯化

利用基因重组技术 ,构建成人肝刺激因子 (hHSS)和谷胱甘肽转移酶 (GST)融合表达载体 ,转化大肠杆菌BL 2 1(DE3 ) ,以His·Tag亲和层析纯化表达产物 ,FactorXa切割分离hHSS单体 ,并检测其生物学活性。结果显示 ,在pET 4 2a表达体系中hHSS以可溶性蛋白和包涵体两种形式存在 ,GST hHSS表达量占菌体可溶性蛋白的3 0 % ;FactorXa切割GST与hHSS之间肽腱 ,得到 3 3和 15kD两条蛋白带 ,经Western杂交证实 3 3kD条带为GST ,而 15kD条带的分子量与hHSS基因序列推测蛋白结果相符。经His·Tag再次纯化可获得hHSS单体 ,初步证实重组hHSS具有促进肝癌细胞增殖活性     

猪繁殖与呼吸综合征病毒非结构蛋白NSP2的原核表达及其单克隆抗体的研制

将猪繁殖与呼吸综合征病毒(PRRSV)S1株NSP2蛋白基因进行截短修饰 (tNSP2),克隆于pGEX-6P-1载体,转化大肠杆菌后用IPTG诱导表达。Western-blot结果表明,融合表达的GST-tNSP2蛋白能被PRRSV阳性血清特异性识别,大小约50kD。经GST柱提取纯化蛋白GST- tNSP2,免疫BALB/c小鼠,取脾细胞与SP2/0骨髓瘤细胞进行融合,获得2株能稳定分泌抗NSP2蛋白抗体的杂交瘤细胞株,将其命名为2B5、3H3。亚型鉴定结果均为IgG 1型,其轻链均为Κ链。间接免疫荧光试验证明,2B5和3H3均能与PRRSV S1毒株产生特异性反应,而不能与SY0608毒株反应。从而为PRRSV分离株的鉴定及NSP2功能研究奠定了重要基础。     

猪链球菌2型mrp基因免疫功能片段的克隆、表达及动物试验

根据猪链球菌2型（Streptococcus suis type 2）国外分离株的溶菌酶释放蛋白（Muramidasereleased protein, MRP）的基因序列,设计并合成一对引物,利用PCR技术扩增了江苏分离株的开放阅读框298～827bp间529bp的基因片段,并定向克隆至pET32a(+)表达载体中。重组质粒经限制性酶切鉴定和测序,转化至大肠杆菌BL21,经IPTG诱导,可表达分子量约42kD的蛋白。经过镍亲和层析柱层析,获得纯化的重组蛋白。以重组蛋白免疫Balb/c小鼠,以5LD50猪链球菌强毒株攻击后小鼠的相对存活率达625%。证实所表达的MRP片段为重要的保护性抗原。     

水稻锌指蛋白OsRZ3基因克隆及融合蛋白的原核表达、纯化

根据NCBI登录的水稻锌指蛋白基因(NO.AK062094,OsRZ3 gene)设计特异引物,通过RT-PCR克隆该基因cDNA全长序列,核苷酸测序并比对氨基酸同源性表明具有C2HC-锌指结构域、分析预测蛋白质分子量为34kD和等电点为9.05;拟南芥原生质体亚细胞定位检测表明其在细胞核。构建pGEX6P-3::OsRZ3融合载体,电转化的大肠杆菌BL21(DE3)菌株经1 mmol·L-1IPTG小量诱导表达,SDS-pAGEX蛋白电泳表明60 kD融合蛋白4 h最大;在LB液体培养中0.5 mmol·L-1IPTG过夜大量诱导表达,通过GST吸附柱层析获得大量包涵体纯化蛋白,1L菌液可诱导纯化到浓度1.58 mg·mL-1包涵体融合蛋白。所构建的融合蛋白原核表达系统能有效表达pGEX6P-3::OsRZ3融合蛋白,0.5 mmol·L-1IPTG大量诱导,通过GST柱吸附获得包涵体纯化蛋白,为作进一步的抗体制备和染色质免疫共沉淀等DNA结合的特性研究准备了基础。     

GST—talinl融合蛋白的原核表达及纯化

应用PCR方法扩增talinl的cDNA,并将其重组入谷胱甘肽转硫酶融合基因表达载体pGEX-4T-1中,获取人源的GST—talinl融合蛋白,为下阶段深入的研究talinl的结构、功能、及其与之相互作用的蛋白打下基础。经酶切、序列鉴定．选择正确重组子,将其质粒转化大肠杆菌BL21（DE3）,IPTG诱导表达,用Glutathione Sepharose 4B柱纯化,western blot鉴定。克隆得到了一个2400bp的talinl的cDNA片断,重组质粒目的DNA测序正确,纯化出分子量约为121、6kD的融合蛋白。用基因工程方法使GST—talinl重组质粒在原核细胞表达并成功纯化出GST—talinl融合蛋白。     

核糖核酸酶抑制因子(RI)的融合表达与复性

应用PCR技术从核糖核酸酶抑制因子 (ribonucleaseinhibitor ,RI)的克隆载体pT7 ri中扩增出ri片段 (1 5kb) ,亚克隆到融合表达载体pGEX 2T中 ,并转化感受态大肠杆菌BL2 1.异丙基半乳糖苷 (IPTG)诱导表达的GST RI经SDS PAGE证明分子量约 76kD ,表达量约占菌体蛋白总量 2 0 % .以包涵体形式表达的目的蛋白经尿素变性 ,透析复性得到的产物具有较高的抑制RNaseA的活性(15 0U ml) .复性的融合蛋白于 2 4℃经凝血酶作用 16h ,可被切割成 5 0kD的RI和 2 6kD的GST .     

人GST-AWP1融合蛋白的原核表达及其抗体制备

为进一步研究人的一新蛋白———蛋白激酶C相关激酶 1相关蛋白 (AWP1)的结构、功能及与其相互作用的蛋白而进行GST AWP融合蛋白表达载体的构建、原核表达、纯化及其抗体的制备 .采用逆转录PCR(RT PCR)法从人ECV30 4内皮细胞中扩增AWP1cDNA编码区 ,并将其重组于谷胱甘肽硫转移酶 (GST)融合蛋白表达质粒pGEX KG中 .经酶切、序列鉴定分析后 ,用该重组质粒转化大肠杆菌BL2 1,并经异丙基 β D 硫代半乳糖苷 (IPTG)诱导产生GST AWP1融合蛋白 ,继而纯化获得了分子量约 5 6kD的融合蛋白 .将此融合蛋白免疫新西兰兔 ,经ELISA和Western印迹检测获得了效价高、免疫活性强的兔抗人多克隆抗体 .结果表明成功构建了GST AWP1融合蛋白表达载体 ,在大肠杆菌高效表达了GST AWP1融合蛋白 ,并获得高效多抗 ,为下阶段深入AWP1功能研究提供了重要的基础     

乙型肝炎病毒DNA聚合酶反式调节蛋白HBVDNAPTP1的表达、纯化与初步鉴定

目的构建HBVDNAPTP1基因的原核表达载体,诱导其在大肠埃希菌中表达,并对融合蛋白进行纯化。方法利用逆转录-PCR获得乙型肝炎病毒(HBV)DNA聚合酶(Polymerase)反式调节人类新基因HBVD-NAPTP1,测序正确后插入至原核表达载体pET-32a(+)中,转化BL21(DE3)宿主菌进行诱导,并利用组氨酸亲和层析方法对融合蛋白进行纯化。结果 HBVDNAPTP1原核表达载体转化宿主菌后,经0.5 mmol/L IPTG、30℃诱导5 h获得了分子量约为31 kD的HBVDNAPTP1融合蛋白的优化表达,Western blotting证实融合蛋白的特异性。亲和层析纯化后得到较纯的HBVDNAPTP1融合蛋白,每升培养菌液中可获得2.24 mg的纯化蛋白。结论成功获得纯化的HBVDNAPTP1融合蛋白,为今后开展HBVDNAPTP1的生物学功能研究奠定了物质基础。     

肿瘤相关抗原基因HCA520重组蛋白的表达纯化及其肝癌患者血清中相应抗体的分析

HCA5 2 0是用SEREX(serologicalidentificationofrecombinantcDNAexpressingcloning)方法 ,即用肝癌病人血清从肝癌组织cDNA表达文库中筛选得到的肝癌相关性抗原编码基因 .利用RT PCR技术检测了HCA5 2 0mRNA在各种组织中的分布情况 ,构建了GST融合表达载体并且用亲和层析的方法纯化表达的融合蛋白 .最后用Western印迹检测了重组蛋白的免疫反应性 ,用斑点印迹检测了肝癌病人血清中HCA5 2 0的天然抗体的存在情况 .结果表明 ,HCA5 2 0在各种组织中呈丰度差异分布 ,构建好的pGEX 4T 3重组载体经IPTG诱导后高效表达GST HCA5 2 0融合蛋白 ,其分子量约 4 9kD .经GST Agarose亲和层析 ,重组蛋白得到高度纯化 .Western印迹证实 ,纯化蛋白为目的重组蛋白 ,重组蛋白具有与天然蛋白相同或相似的免疫反应性 .斑点印迹分析表明 ,2 0份肝癌病人血清中有 1份HCA5 2 0抗体阳性 ,而 4份正常人均为阴性 .HCA5 2 0的足量提供 ,可用以研究其在致癌中的作用 ,并可以免疫动物制备抗体 .它作为抗原 ,分析其抗体在不同肿瘤病人中的表达情况 ,评估其在临床肿瘤诊断中的作用     

游仆虫Rab蛋白基因的克隆表达与纯化

为对单细胞原生动物纤毛虫中Rab蛋白的功能进行研究 ,进而探讨以胞吞和胞吐为主要物质交换途径的纤毛虫中囊泡定向运输的机理 .利用PCR技术从游仆虫大核DNA及cDNA中扩增出rab基因 ,并进行了序列分析 ,该基因全长为 783bp ,两端为端粒序列 ,编码框为 6 2 4bp ,编码 2 0 7个氨基酸 ,开放读框中有 3个TGA ,在此编码半胱氨酸 .利用定点突变将rab基因中 3个TGA突变为通用半胱氨酸密码子TGC .将游仆虫Rab蛋白基因构建于原核表达载体pGEX 4T 2中 ,得到的重组质粒pGEX Eorab1转化至大肠杆菌BL2 1(DE3)中 ,IPTG诱导表达 .表达产物与抗GST抗体在 4 9kD处有很强的交叉反应 .融合蛋白GST EoRab1通过亲和层析柱纯化和凝血酶的切割 ,再经两步纯化得到电泳纯的游仆虫Rab蛋白 .     

Design and characterization of viral polypeptide inhibitors targeting Newcastle disease virus fusion

Paramyxovirus infections can be detected worldwide with some emerging zoonotic viruses and currently there are no specific therapeutic treatments or vaccines available for many of these diseases. Recent studies have demonstrated that peptides derived from the two heptad repeat regions (HR1 and HR2) of paramyxovirus fusion proteins could be used as inhibitors of virus fusion. The mechanism underlying this activity is in accordance with that of class I virus fusion proteins, of which human immunodeficiency virus (HIV) and influenza virus fusion proteins are members. For class I virus fusion proteins, the HR1 fragment binds to HR2 to form a six-helix bundle with three HR1 fragments forming the central coiled bundle surrounded by three coiled HR2 fragments in the post fusion conformational state (fusion core). It is hypothesized that the introduced exogenous HR1 or HR2 can compete against their endogenous counterparts, which results in fusion inhibition. Using Newcastle disease virus (NDV) as a model, we designed several protein inhibitors, denoted HR212 as well asHR121 and 5-Helix, which could bind the HR1 or HR2 region of fusion protein, respectively. All the proteins were expressed and purified using a GST-fusion expression system in Escherichia coli. The HR212 or GST-HR212 protein, which binds the HR1 peptide in vitro, displayed inhibitory activity against NDV-mediated cell fusion, while the HR121 and 5-Helix proteins, which bind the HR2 peptide in vitro, inhibited virus fusion from the avirulent NDV strain when added before the cleavage of the fusion protein. These results showed that the designed HR212, HR121 or 5-Helix protein could serve as specific antiviral agents. These data provide additional insight into the difference between the virulent and avirulent strains of NDV.     

腮腺炎病毒F蛋白七肽重复序列的表达、纯化及特性分析

副粘病毒F蛋白的两段七肽重复序列（HR1和HR2）在病毒侵染细胞的过程中相互作用形成热稳定的富含α螺旋的异源二聚体,此结构的形成引起病毒囊膜与细胞膜的并置而最终导致膜融合的发生。腮腺炎病毒（Mumps virus, MuV）属于副粘病毒科,腮腺炎病毒属,可能利用与其他副粘病毒相似的侵染机制。本研究对MuV 融合蛋白的HR区进行了计算机程序预测,并利用大肠杆菌GST融合表达系统对MuV F蛋白HR1和HR2两段多肽进行了表达和纯化,通过GST pull_down 实验证实HR1和HR2多肽在体外能够相互作用,凝胶过滤层析证明HR1、HR2多肽能够形成多聚体,说明MuV F蛋白的HR区的相互作用可能是其发挥融合功能的关键因素。     

新城疫病毒3种检测方法的研究比较

目的:利用3种方法对新城疫(Newcastle disease virus, NDV)病毒进行检测并对这3种检测方法的优缺点做出比较。方法:分别将NDV强毒F48E9和弱毒Lasota接种SPF鸡胚后,获取尿囊液。利用双抗夹心ELISA法、悬液芯片系统以及RT-PCR进行检测。通过对制备的针对新城疫病毒的抗体4D9和6C4蛋白浓度测定后,选择6C4进行生物素标记,将4D9作为固相捕获抗体,利用生物素-链霉亲和素放大系统构建双抗夹心检测体系。通过对Genebank上已发表的新城疫强弱毒F基因进行电脑分析后,设计一组针对NDV强弱毒的通用型引物,分别对强弱毒进行RT-PCR并检测其检出限。结果:ELISA法对NDV强弱毒尿囊液的检出灵敏度为1:160,但操作繁琐,耗时长;液相芯片对强弱毒尿囊液的检出限为1:160和1:320,然而和ELISA相比,操作较为方便,但仪器设备昂贵。RT-PCR对强弱毒RNA检出限分别为259pg和14pg,与前两种方法相比,PR-PCR在核酸水平上对病毒进行检测,理论上灵敏度较高,但是所需试剂、设备昂贵,且实验人员还需一定的技能培训。     

Comparison of substrate specificities against the fusion glycoprotein of virulent Newcastle disease virus between a chick embryo fibroblast processing protease and mammalian subtilisin-like proteases

The fusion (F) protein precursor of virulent Newcastle disease virus (NDV) strains has two pairs of basic amino acids at the cleavage site, and its intracellular cleavage activation occurs in a variety of cells; therefore, the viruses cause systemic infections in poultry. To explore the protease responsible for the cleavage in the natural host, we examined detailed substrate specificity of the enzyme in chick embryo fibroblasts (CEF) using a panel of the F protein mutants at the cleavage site expressed by vaccinia virus vectors, and compared the specificity with those of mammalian subtilisin-like proteases such as furin, PC6 and PACE4 which are candidates for F protein processing enzymes. It was demonstrated in CEF cells that Arg residues at the -4, -2 and -1 positions upstream of the cleavage site were essential, and that at the -5 position was required for maximal cleavage. Phe at the +1 position was also important for efficient cleavage. On the other hand, furin and PC6 expressed by vaccinia virus vectors showed cleavage specificities against the F protein mutants consistent with that shown by the processing enzyme of CEF cells, but PACE4 hardly cleaved the F proteins including the wild type. These results indicate that the proteolytic processing enzymes of poultry for virulent NDV F proteins could be furin and/or PC6 but not PACE4. The significance of individual contribution of the three amino acids at the -5, -2 and +1 positions to cleavability was discussed in relation to the evolution of virulent and avirulent NDV strains.     

Generation by Reverse Genetics of an Effective,Stable, Live-Attenuated Newcastle Disease Virus Vaccine Based on a Currently Circulating,Highly Virulent Indonesian Strain

Newcastle disease virus (NDV) can cause severe disease in chickens. Although NDV vaccines exist, there are frequent reports of outbreaks in vaccinated chickens. During 2009–2010, despite intense vaccination, NDV caused major outbreaks among commercial poultry farms in Indonesia. These outbreaks raised concern regarding the protective immunity of current vaccines against circulating virulent strains in Indonesia. In this study, we investigated whether a recombinant attenuated Indonesian NDV strain could provide better protection against prevalent Indonesian viruses. A reverse genetics system for the highly virulent NDV strain Banjarmasin/010/10 (Ban/010) isolated in Indonesia in 2010 was constructed. The Ban/010 virus is classified in genotype VII of class II NDV, which is genetically distinct from the commercial vaccine strains B1 and LaSota, which belong to genotype II, and shares only 89 and 87% amino acid identity for the protective antigens F and HN, respectively. A mutant virus, named Ban/AF, was developed in which the virulent F protein cleavage site motif “RRQKR↓F” was modified to an avirulent motif “GRQGR↓L” by three amino acid substitutions (underlined). The Ban/AF vaccine virus did not produce syncytia or plaques in cell culture, even in the presence of added protease. Pathogenicity tests showed that Ban/AF was completely avirulent. Ban/AF replicated efficiently during 10 consecutive passages in chickens and remained genetically stable. Serological analysis showed that Ban/AF induced higher neutralization and hemagglutination inhibition antibody titers against the prevalent viruses than the commercial vaccines B1 or LaSota. Both Ban/AF and commercial vaccines provided protection against clinical disease and mortality after challenge with virulent NDV strain Ban/010 (genotype VII) or GB Texas (genotype II). However, Ban/AF significantly reduced challenge virus shedding from the vaccinated birds compared to B1 vaccine. These results suggest that Ban/AF can provide better protection than commercial vaccines and is a promising vaccine candidate against NDV strains circulating in Indonesia.     

精制狂犬病疫苗纯化方法的比较研究

地鼠肾细胞狂犬病疫苗原液经１００ ｋＤ 膜浓缩 ３０ 倍,分别选用（１）ＤＥＡＥ Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ离子交换层析法;（２）Ｓｅｐｈａｃｒｙ１ Ｓ－２００ ＨＲ 分子筛选层析法;（３）二次蔗糖等密度区带离心法对其进行纯化。用此三种方法各试制３ 批精制疫苗,结果表明,经ＤＥＡＥ Ｓｅｐｈａｒｏｓｅ ＣＬ－６Ｂ离子交换层析纯化后疫苗总蛋白含量减少９９％ 以上,抗原比活性提高１５９ 倍,抗原回收率达５０％ ,纯化疫苗以ＮＩＨ 法效力测定平均为５．４ ＩＵ／２ｍ ｌ;经Ｓｅｐｈａｃｒｙ１ Ｓ－２００ＨＲ 分子筛层析纯化后疫苗总蛋白含量减少 ９８％ 以上,抗原比活性提高４１ 倍,抗原回收率达６３％ ,纯化疫苗效力平均为６．２５ ＩＵ／２ｍ ｌ;经一次蔗糖等密度区带离心法纯化后疫苗总蛋白含量减少９８％ 以上,抗原比活性提高３２１ 倍,抗原回收率达４３％ ,纯化疫苗效力平均为６．１８ ＩＵ／２ｍ ｌ,三种纯化疫苗均符合Ｗ ＨＯ 规程要求。     

The hemagglutinin-neuraminidase protein of Newcastle disease virus determines tropism and virulence

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a crucial role in the process of infection. However, the exact contribution of the HN gene to NDV pathogenesis is not known. In this study, the role of the HN gene in NDV virulence was examined. By use of reverse genetics procedures, the HN genes of a virulent recombinant NDV strain, rBeaudette C (rBC), and an avirulent recombinant NDV strain, rLaSota, were exchanged. The hemadsorption and neuraminidase activities of the chimeric viruses showed significant differences from those of their parental strains, but heterotypic F and HN pairs were equally effective in fusion promotion. The tissue tropism of the viruses was shown to be dependent on the origin of the HN protein. The chimeric virus with the HN protein derived from the virulent virus exhibited a tissue predilection similar to that of the virulent virus, and vice versa. The chimeric viruses with reciprocal HN proteins either gained or lost virulence, as determined by a standard intracerebral pathogenicity index test of chickens and by the mean death time in chicken embryos (a measure devised to classify these viruses), indicating that virulence is a function of the amino acid differences in the HN protein. These results are consistent with the hypothesis that the virulence of NDV is multigenic and that the cleavability of F protein alone does not determine the virulence of a strain.     

表达新城疫病毒F48E8株融合蛋白基因的重组鸡痘病毒及其免？…

将将城疫病毒（ＮＤＶ）Ｆ４８Ｅ８株融合蛋白基因导入鸡痘病毒（ＦＰＶ）插入载体ｐＥＧＦ１１７５－１的Ｐ７．５启动子下游,得到转移载体ｐＦＧ１１７５－１重组质粒。采用脂质体转染技术,将该质粒转染ＦＰＶ２８２Ｅ株感染的鸡胚成纤维细胞（ＣＥＦ）。,经过多次蓝斑筛选纯化,获稳定的重组病毒ｒＦＰＶ－ＮＤＦ。间接免疫荧光试验表明,ｒＦＰＶ－ＮＤＦ感染的ＣＥＦ中表达了ＮＤＶ的融合蛋白。用ｒＦＰＶ－ＮＤＦ免疫的ＳＦ     

鲍曼不动杆菌烈性噬菌体的分离与纯化

利用柱层析方法,纯化鲍曼不动杆菌（Acinetobacter baumannii）烈性噬菌体AB1。首先采用聚乙二醇6000沉淀方法,初步分离裂解液中的噬菌体,噬菌体纯度由6.1×1010 pfu/mg提高到37×1010 pfu/mg,噬菌体回收率为58.8%,蛋白质去除率为90.6%;噬菌体粗提样品经Sepharose 4B凝胶过滤层析柱进一步纯化,纯度提高到73×1010 pfu/mg,噬菌体回收率为95.7%,蛋白质去除率为48.1%;收集的噬菌体样品最后经DEAE-52阴离子交换层析柱处理,噬菌体纯度为40×1010 pfu/mg,回收率为50.8%,蛋白去除率15.6%。内毒素分析结果显示,Sepharose 4B凝胶过滤层析纯化的噬菌体样品中,内毒素含量为443.8 EU/mg,而DEAE-52阴离子交换层析纯化的噬菌体样品中,内毒素含量为544.4 EU/mg。实验结果显示,PEG沉淀方法与Sepharose 4B凝胶过滤方法能够有效地提高噬菌体纯度,而DEAE-52阴离子交换层析则不能提高噬菌体的纯度,也无法有效地去除样品中的内毒素。     

Genetic Diversity of Newcastle Disease Virus in Wild Birds and Pigeons in West Africa

In West and Central Africa, virulent Newcastle disease virus (NDV) strains of the recently identified genotypes XIV, XVII, and XVIII are enzootic in poultry, representing a considerable threat to the sector. The increasing number of reports of virulent strains in wild birds at least in other parts of the world raised the question of a potential role of wild birds in the spread of virulent NDV in sub-Saharan Africa as well. We investigated 1,723 asymptomatic birds sampled at live-bird markets and sites important for wild-bird conservation in Nigeria and 19 sick or dead wild birds in Côte d''Ivoire for NDV class I and II. Typical avirulent wild-type genotype I strains were found in wild waterfowl in wetlands in northeastern Nigeria. They were unrelated to vaccine strains, and the involvement of inter- or intracontinental migratory birds in their circulation in the region is suggested. Phylogenetic analyses also revealed that genotype VI strains found in pigeons, including some putative new subgenotype VIh and VIi strains, were introduced on multiple separate occasions in Nigeria. A single virulent genotype XVIII strain was found in a dead wild bird in Côte d''Ivoire, probably as a result of spillover from sick poultry. In conclusion, screening of wild birds and pigeons for NDV revealed the presence a variety of virulent and avirulent strains in West Africa but did not provide strong evidence that wild birds play an important role in the spread of virulent strains in the region.