Prokaryotic expression and potential application of the truncated PCV-2 capsid protein

Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 (PCV-2). The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a(+) vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta(DE3) E. coli and expressed by induction of IPTG. Expressed proteins were detected by western blotting and ELISA. The protein with best immunoreactivity was confirmed and selected, then utilized to inoculate SPF rabbits to prepare polyclonal antibodies. The protein and prepared polyclonal antibody were utilized to detect sera samples against PCV-2 from Shandong province and PCV-2 particles in PK-15 cells. In our study, three recombinant fusion proteins were successfully obtained, and the molecular weights of fusion proteins were 35.9 kDa, 33.6 kDa and 38.6 kDa respectively detected by SDS-PAGE. All of the proteins showed positive reaction with anti-PCV-2 antisera, and His-XF2-2 showed better immunoreactivity than the others. The protein of His-XF2-2 was coated as antigen in ELISA to detect the seroprevalence of PCV-2 in certain districts of Shandong province, the seropositivity rate was 27.7 % (73/264). Specific fluorescence and positive signals for PCV-2 could be detected in PK-15 cells inoculated with PCV-2 with the participation of prepared antibodies against His-XF2-2 in IFA and IPMA. Experimental results indicated that the truncated PCV-2 ORF2 gene containing most of the NLS region was successfully expressed in E. coli, and His-XF2-2 was demonstrated to have better immunoreactivity with anti-PCV-2 antisera than the other two fusion proteins. His-XF2-2 and prepared polyclonal antibodies against it had a satisfactory capability in detecting PCV-2 infection.     

猪圆环病毒2型ORF2编码基因真核表达载体的构建及表达

根据GenBank中猪圆环病毒2型(PCV-2)ORF2基因序列,设计了1对引物,应用PCR从含有PCV-2的PK-15细胞中扩增出ORF2基因,将其克隆入pSecTag2载体中,构建了pSecORF2载体。又设计一条含信号肽序列的上游引物,以pSecORF2为模板,扩增出含信号肽序列的ORF2基因,将其克隆到pIREShyg载体上,构建了pIRESiORF2真核表达载体。然后通过磷酸钙共沉淀法转染CHO细胞,进行表达。间接免疫荧光实验(IFA)成功检测到pIRESiORF2在CHO细胞中的表达。这为进一步研究ORF2编码蛋白的生物学活性及建立PCV诊断试剂盒打下基础。     

鸡贫血病毒衣壳蛋白的表达纯化及多克隆抗体制备

利用PCR技术,以pPrpo-VP1为模板扩增得到鸡贫血病毒的衣壳蛋白基因(VP1),以T4多聚核苷酸激酶磷酸化处理、纯化后,克隆至表达载体pET-30a( )中,从而构建了原核表达质粒pET30-VP1。将pET30-VP1转化至感受态细胞E.coliBL21(DE3)中,经IPTG诱导后,SDS-PAGE分析,可见约45kDa的目的蛋白获得表达。该蛋白经亲和层析纯化后,免疫6-8w的雌性Balb/c鼠,三次免疫后,采血分离血清,制得抗VP1的多克隆血清。以纯化的VP1为包被抗原,用ELISA方法检测,制备的血清效价达12800×以上。以Westernblot检测,该血清可与目的蛋白发生特异性反应,证明其具有良好的免疫原性。VP1蛋白的成功表达及其多克隆抗体的制备为进一步研究VP1蛋白的功能及开展CAV疫苗及诊断制剂的研制奠定了基础。     

鸡贫血病毒衣壳蛋白的表达纯化及多克隆抗体制备

利用PCR技术,以pPrpo-VP1为模板扩增得到鸡贫血病毒的衣壳蛋白基因(VP1),以T4多聚核苷酸激酶磷酸化处理、纯化后,克隆至表达载体pET-30a(+) 中,从而构建了原核表达质粒pET30-VP1.将pET30-VP1转化至感受态细胞E.coli BL21(DE3)中,经IPTG诱导后,SDS-PAGE分析,可见约45kDa的目的蛋白获得表达.该蛋白经亲和层析纯化后,免疫6-8w的雌性Balb/c鼠,三次免疫后,采血分离血清,制得抗VP1的多克隆血清.以纯化的VP1为包被抗原,用ELISA方法检测,制备的血清效价达12800×以上.以Western blot 检测,该血清可与目的蛋白发生特异性反应,证明其具有良好的免疫原性.VP1蛋白的成功表达及其多克隆抗体的制备为进一步研究VP1蛋白的功能及开展CAV疫苗及诊断制剂的研制奠定了基础.     

猪细小病毒VP2蛋白的主要抗原表位基因的原核表达及检测应用

将构建好的重组质粒PET-VP2Ⅰ转化宿主菌BL21,利用IPTG(1mmol/L)诱导实现了PET-VP2Ⅰ蛋白主要抗原域VP2Ⅰ融合蛋白的高效表达。通过Western-blot检测证明表达的重组蛋白具有良好的免疫学活性。表达产物经HisBind层析柱纯化后作为诊断抗原初步建立了检测PPV抗体的间接ELISA方法。结果表明:抗原的最佳包被浓度为3.5μg/mL,血清的最佳稀释度为1∶40,待检血清阳性标准初步定为OD490>0.51,且待检血清的OD490值与阴性血清的OD490值的比值大于2.1。     

铁蛋白与法氏囊病毒核衣壳蛋白重组蛋白的原核表达及纳米颗粒胞外自组装

传染性法氏囊病毒（infectious bursal disease virus,IBDV）是传染性法氏囊病（infectious bursal disease,IBD）的病原,其导致的高死亡率使全球的家禽养殖业受到了严重的经济损失。近年来,基因工程疫苗和DNA疫苗的迅速发展为IBD更有效的防控带来了希望。基于自然合成的铁蛋白大多可以组装成较稳定的多聚体这一特点,其可以作为很多疾病的免疫原载体。根据大肠杆菌密码子的偏好性,对IBDV-VP2、铁蛋白序列进行了优化,并通过大肠杆菌表达系统进行了融合表达,通过质谱、Western blotting、SDS-PAGE检测,该融合蛋白已成功在大肠杆菌中以包涵体的形式表达,且通过透射电镜可观察到大小均一的纳米级颗粒,研究结果为后期探究铁蛋白自组装对提高IBDV疫苗的广谱性、免疫原性的作用提供了参考,也为进一步研究其作为药物和抗原载体的应用机制奠定了基础。     

猪圆环病毒2型的PCR检测方法应用

本研究登录Genbank对猪圆环病毒2型基因的序列进行分析,用Primer 5.0设计了ORF2基因的扩增引物,试图选择一种比较合理的PCR方法检查PCV2感染的病原,以期这种PCR方法可以有效分析猪综合征障碍病毒(PRRSV)、猪细小病毒(PPV)、猪瘟病毒的扩增(HCV)、猪伪狂犬病毒(PRV)等比较常见的病原。研究结果表明,在PCV2进行扩增阳性样品检测中,发现一条异常447 bp的DNA条带,对产物测序结果进行扩增分析,证明其是PCV ORF2基因序列。敏感性检验分析表明检测样本DNA浓度时达到了9.8×10^-4ng/μL。PCR实验具有良好的稳定性和重复性。根据对66例临床病猪的分析表明,在PCV2的检测中阳性感染率是27.26%,这可能受到HCV、PRRSV、PRV、PPV等多种病毒的感染,其中混合感染的比例达到了72.23%。     

人Nek2蛋白原核表达纯化及其多克隆抗体制备 *

目的通过原核表达系统表达人Nek2蛋白,优化表达条件并纯化Nek2蛋白,制备抗Nek2多克隆抗体.方法 将Nek2基因片段构建到原核表达载体pET30a(+)上,转化大肠杆菌BL21 (DE3);加入诱导剂IPTG诱导表达,对诱导温度,诱导剂IPTG终浓度,诱导时间等条件进行优化;利用12% SDS-PAGE后250mmol/L KCl染色切胶纯化蛋白质,将纯化后的Nek2蛋白进行质谱鉴定;纯化Nek2蛋白免疫BALB/c小鼠制备多克隆抗体,运用ELISA,Western blot和免疫荧光实验检测多克隆抗体效价和特异性.结果 构建了pET30a(+)-Nek2重组原核表达质粒,诱导的重组人Nek2蛋白主要以包涵体的形式存在;蛋白质的最适诱导表达条件为28℃,180r/min条件下加入终浓度为0.2mmol/L IPTG诱导32h;质谱分析纯化后的蛋白质为Nek2蛋白,最终获得浓度为1.35mg/ml纯化后的Nek2蛋白;纯化蛋白免疫小鼠,多克隆抗体效价大于1∶243 000,且具有良好的抗原特异性;免疫荧光实验显示Nek2主要定位于细胞质和细胞核.结论: 利用重组人Nek2蛋白获得具有良好抗原特异性的抗Nek2多克隆抗体.     

cTnI-linker-TnC融合蛋白的原核表达及鉴定

从PUBMED中查找人心脏cTnI和cTnC的cDNA序列,在两者之间加上15个中性氨基酸残基(G4S)3的linker编码序列,并分别构建原核表达质粒pET32a-cTnI-linker-TnC和pET28a-cTnI-linker-TnC,转化入大肠杆菌表达菌株BL21(DE3)中,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导其表达。通过SDS-PAGE电泳和间接ELISA对目的蛋白进行初步鉴定,并优化表达条件以实现目的蛋白的可溶性高表达。用标准抗cTnI单克隆抗体和标准抗cTnC单克隆抗体对目的蛋白进行Western blot鉴定和双抗体夹心ELISA鉴定,同时用八位心肌梗塞患者血清做间接ELISA鉴定。结果表明,cTnI-linker-TnC融合蛋白在表达载体pET32a中实现了可溶性高表达,最佳表达条件为28℃、0.4mmol/L IPTG、诱导表达5h;且融合蛋白具有较好的免疫原性,保存了cTnI和cTnC单体的天然结构,有望成为天然cTnI-TnC复合物的替代物,为下一步制备高特异性的抗cTnI-TnC复合物的单克隆抗体奠定了基础。     

何首乌中Ⅲ型聚酮合酶基因FmPKS2的原核表达及鉴定

目的：对传统中药何首乌中Ⅲ型聚酮合酶基因FmPKS2进行原核表达并鉴定重组蛋白酶活性,为研究该酶基因在何首乌有效成分代谢合成及其调控中的功能奠定基础。方法：根据何首乌FmPKS2（GenBank登录号：GQ984139）基因序列,通过PCR扩增其全长的编码区,克隆至原核表达载体PET-28a,构建重组质粒PET-FmPKS2,将其转化原核表达菌株E.coli BL21（DE3）,并用IPTG诱导表达,SDS-PAGE鉴定融合蛋白可溶性,通过Ni-NTA亲和树脂纯化可溶性蛋白后以丙二酰-COA和香豆酰-COA为底物进行催化反应,TLC鉴定反应产物。结果：经过诱导,重组菌表达分子量为42KD左右的融合蛋白,其中可溶性重组蛋白最优表达条件为IPTG浓度0.5 mmol/L,诱导时间6h,温度25℃。纯化后的可溶性重组蛋白催化丙二酰-COA和香豆酰-COA得到的产物经TLC鉴定为二苯乙烯类化合物白藜芦醇。结论：成功实现FmPKS2的原核表达且融合蛋白以可溶形式存在,催化反应证明FmPKS2融合蛋白具有二苯乙烯合酶的活性。     

猪圆环病毒2型福建株的全基因组克隆与序列分析

根据GenBank上公布的猪圆环病毒2型全基因序列设计一对扩增PCV-2全基因的引物,建立扩增PCV-2全序列的PCR方法,并应用此方法从福建省不同地区采集的疑似断奶仔猪多系统衰竭综合征(PMWS)的仔猪肺组织病料中扩增出PCV-2全基因组(1 767 bp),将此基因片段克隆入pMD 18-T载体,筛选获得重组质粒pMD-PCV-2,并对其进行序列测定,然后对全基因组进行同源性和遗传进化分析。结果表明,福建省不同地区采集的猪肺组织扩增出的PCV-2全序列与GenBank上公布的的全基因组同源性介于94.9%-99.8%之间,ORF1的同源性介于97.2%-99.9%,ORF2的同源性也很高,介于97.7%-99.8%之间。其中福州株、福清株和漳州株与中国农大报道的12株、郑州株5株、杭州4株、武汉株3株、上海株2株、扬州2株、南京1株和兰州1株共30株在一个进化分支上,同源性也高达99.3%。本研究有助于监测PCV-2的疫源和进化关系,为进一步深入研究福建省生猪猪圆环病毒来源奠定一定基础。     

Development of a loop-mediated isothermal amplification assay for porcine circovirus type 2

In this study,the loop-mediated isothermal amplification(LAMP)method was used to develop a rapid and simple detection system for porcine circovirus type 2(PCV2).According to the PCV2 sequences published in GenBank,multiple LAMP primers were designed targeting conserved sequences of PCV2.Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template,LAMP reactions in a PCV2 LAMP system was performed,the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye.The results showed highly-efficient and specific amplification in 30 min at 63℃ with a LAMP real-time turbidimeter.Furthermore,PCV2 DNA templates,with a detection limit of 5.5×10-5ng of nucleic acid,indicated that this assay was highly sensitive.The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter,showing the potential simplicity of interpretation of the assay results.The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples.In addition it offers higher specificity and sensitivity,shorter reaction times and simpler procedures than the currently available methods of PCV2 detection.It is therefore a promising tool for the effective and efficient detection of PCV2.     

Protective immunity conferred by porcine circovirus 2 ORF2‐based DNA vaccine in mice

Post‐weaning multisystemic wasting syndrome (PMWS) associated with porcine circovirus type 2 (PCV2) has caused the swine industry significant health challenges and economic damage. Although inactivated and subunit vaccines against PMWS have been used widely, so far no DNA vaccine is available. In this study, with the aim of exploring a new route for developing a vaccine against PCV2, the immunogenicity of a DNA vaccine was evaluated in mice. The pEGFP‐N1 vector was used to construct a PCV2 Cap gene recombinant vaccine. To assess the immunogenicity of pEGFP‐Cap, 80 BALB/c mice were immunized three times at 2 weekly intervals with pEGFP‐Cap, LG‐strain vaccine, pEGFP‐N1 vector or PBS and then challenged with PCV2. IgG and cytokines were assessed by indirect ELISA and ELISA, respectively. Specimens stained with hematoxylin and eosin (HE) and immunohistochemistry (IHC) techniques were examined histopathologically. It was found that vaccination of the mice with the pEGFP‐Cap induced solid protection against PCV2 infection through induction of highly specific serum IgG antibodies and cytokines (IFN‐γ and IL‐10), and a small PCV2 viral load. The mice treated with the pEGFP‐Cap and LG‐strain developed no histopathologically detectable lesions (HE stain) and IHC techniques revealed only a few positive cells. Thus, this study demonstrated that recombinant pEGFP‐Cap substantially alleviates PCV2 infection in mice and provides evidence that a DNA vaccine could be an alternative to PCV2 vaccines against PMWS.     

小麦TaMAPK2激酶基因的原核表达以及多克隆抗体制备

MAPK蛋白激酶是一类重要的植物胁迫信号调控因子.为了研究小麦MAPK基因的功能,苯试验克隆了小麦MAPK蛋白激酶基因TaMAPK2.为了制备TaMAPK2基因的多克隆抗体,TaMAPK2的非保守区段的DNA序列anti-MAPK2被构建到原核表达载体 pET-28a-(+)上,表达融合蛋白His-antiMAPK2.在终浓度为1 mmol/L IPTG诱导1h的条件下,融合蛋白His-antiMAPK2表达量达到最大.通过蛋白标记亲和层析柱(HisTrapTMHP)得到纯化的His-antiMAPK2融合蛋白.利用新西兰大白兔制备了TaMAPK2基因的多克隆抗体,ELISA竞争抑制法检测抗体效价检测,效价为1:80000,能满足后续试验要求的效价值,为进一步分析TaMAPK2的蛋白定位、表达等提供基础.     

Development of a Loop-Mediated Isothermal Amplification Assay for Porcine Circovirus Type 2

In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed di...     

人CD24分子成熟多肽的原核表达及抗体制备

目的: 为获得抗人CD24分子成熟多肽核心蛋白(hCD24N)的多克隆抗体。方法: 制备CD24表达阳性的人肿瘤细胞cDNA,采用PCR法扩增hCD24N的编码基因,构建pGEX-KGV-hCD24N原核表达质粒;转化大肠杆菌BL21(DE3),乳糖诱导表达;经GST亲和柱层析、SDS-PAGE和Western blotting制备并鉴定纯化的GST-StraptagII-hCD24N融合蛋白;免疫新西兰大白兔制备抗血清并用rProtein A亲和柱层析纯化多克隆IgG抗体;用间接ELISA法测定抗体效价,Western blotting鉴定抗体特异性,同时采用细胞免疫荧光检测技术对抗体的特异性和应用可行性作进一步评价。结果: 实现了hCD24N基因的克隆以及在原核细胞中的可溶性重组融合表达,得到了纯化后的目的融合蛋白,并以其为免疫原获得了效价高于1:100 000的抗hCD24N多克隆抗体,Western blotting及细胞免疫荧光检测证明该抗体与当前市售的抗人CD24抗体具有相似的免疫反应特异性,并且能够与CD24阳性人肿瘤细胞表达并加工的高度糖基化CD24天然分子发生特异性抗原-抗体反应。结论: 抗hCD24N多克隆抗体的成功制备为进一步以CD24分子为靶点的肿瘤生物学基础研究以及相关癌症的诊断试剂开发奠定基础。     

SUA41蛋白表达和纯化及其多克隆抗体制备

旨在制备特异性SUA41多克隆抗体,为深入研究其在植物生长发育中的功能提供有力的分子生物学和生物化学的工具。PCR扩增拟南芥SUA41基因中编码280个氨基酸(401-680位氨基酸)的特异片段,经过GATEWAY的DNA重组技术构建了原核表达载体pDEST17-SUA41,用热休克法转化到E.coliBL21(DE3)star感受态细胞,以异丙基β-D-硫代半乳糖苷(IPTG)诱导表达出6×His-SUA41融合蛋白,用8 mol/L尿素缓冲液溶解包涵体并且经过水逐级去除尿素获得提纯的融合蛋白,并利用Western blotting鉴定确认。融合蛋白经Ni金属螯合柱亲和层析得以纯化,用SDS-PAGE进一步纯化。纯化的融合蛋白经过SDS-PAGE后切胶回收,免疫小白兔,制备多抗血清,然后用Western blotting进行检测,鉴定血清特异性和效价。结果显示,融合蛋白6×His-SUA41免疫兔,产生特异性的SUA41兔抗血清,可以检测到细菌和拟南芥组织中SUA41蛋白。用水提纯变性剂尿素溶解的包涵体蛋白具有可行性。制备的特异性SUA41兔抗血清效价高,能够有效地识别大肠杆菌表达的和拟南芥的SUA41蛋白。在有合适的对照情况下,该兔抗血清可以用于分析植物中SUA41蛋白的功能。     

An ELISA based on a truncated soluble ORF2 protein for the detection of PCV2 antibodies in domestic pigs

Postweaning multisystemie wasting syndrome (PMWS) is an important swine disease that is closely associated with porcine circovirus type 2 (PCV2). The capsid protein (Cap protein) is a major structural protein that has at least three immunoreactive regions, and it can be a suitable candidate antigen for detecting the specific antibodies of a PCV2 infection. In the present study, an indirect enzyme-linked immunosorbent assay (TcELISA)based on a truncated soluble Cap protein produced in Escherichia coli (E.coli) was established and validated for the diagnostic PCV2 antibodies in swine. The TcELISA was validated by comparison with an indirect immunofluorescence assay (IIFA). The diagnostic sensitivity (DSN), specificity (DSP), and accuracy of the TcELISA were 88.6%, 90.7% and 89.4%, respectively. The agreement rate was 89.38% between results obtained with TcELISA and IIFA on 113 field sera. A cross-reactivity assay showed that the method was PCV2-specific by comparison with other sera of viral disease. Therefore ,the TcELISA will be helpful for the development of a reliable serology diagnostic test for large scale detection of PCV2 antibodies and for the evaluation of vaccine against PCV2 in swine.     

重组GST-HBx蛋白在表达过程中断裂状况的研究

目的 在对乙型肝炎病毒非结构蛋白HBx研究的过程中,发现体外表达HBx蛋白产物的稳定性存在差异.为了了解其中的原因,拟通过原核表达方式进行分析.方法 以包含全长HBV DNA的质粒pEco63为模板,通过PCR反应,扩增HBV X基因全长序列（1～154氨基酸）和截短序列（48～104氨基酸,48～146氨基酸,98～146氨基酸）,将这几种不同长度和位置的HBX DNA序列克隆到pGEX4T-2原核表达载体中,并通过下游引物设计中引入6×组氨酸标签序列.结果 最终成功构建N端融合GST蛋白标签序列和C端融合6×组氨酸标签序列的4种HBx重组表达载体.将4种包含不同长度HBx的重组质粒转化大肠埃希菌Rosetta,培养至菌液浓度A值大约0.6左右,加入终浓度0.1 mmol/L的IPTG进行诱导表达.提取全菌蛋白通过Western印迹对HBx各重组蛋白产物的N端和C端进行检测.结论 在分段表达的HBx重组蛋白中发生了多处局部的断裂,而断裂部位主要集中于GST蛋白上.而造成这一结果的原因可能与HBx蛋白功能区域暴露后与GST相互作用有关.