V5标记的猪圆环病毒2型Cap蛋白重组杆状病毒表达载体的构建

试验旨在利用真核表达系统构建V5标记的猪圆环病毒2型（PCV2） Cap蛋白的表达载体。采用基因工程技术将V5标签引入到PCV2 ORF2基因C末端,并将标记基因定向克隆入pFastBacHTA载体,将pFastBacHTA-ORF2-V5重组转移载体转化大肠杆菌DH10Bac感受态细胞,使用脂质体介导法将鉴定后的重组杆状病毒质粒转染于Sf9细胞中;运用间接免疫荧光试验（IFA）、SDS-PAGE和Western blotting试验对Sf9细胞中V5标记Cap蛋白的表达进行验证。结果显示,本试验成功将V5标签引入到PCV2 ORF2基因末端,pFastBacHTA-ORF2-V5重组转移载体转化大肠杆菌DH10Bac感受态细胞后获得了Bacmid-ORF2-V5重组杆状病毒质粒;IFA、SDS-PAGE和Western blotting试验结果显示,在Sf9细胞中能检测到重组杆状病毒V5标记的PCV2 Cap蛋白,具有良好的反应原性,说明本试验成功获得一株rAcMNPV Cap-V5重组杆状病毒。试验结果为PCV2标记亚单位疫苗的研制提供一定的理论依据。     

V5标记的猪圆环病毒2型Cap蛋白重组杆状病毒表达载体的构建

试验旨在利用真核表达系统构建V5标记的猪圆环病毒2型(PCV2)Cap蛋白的表达载体。采用基因工程技术将V5标签引入到PCV2 ORF2基因C末端,并将标记基因定向克隆入pFastBacHTA载体,将pFastBacHTA-ORF2-V5重组转移载体转化大肠杆菌DH10Bac感受态细胞,使用脂质体介导法将鉴定后的重组杆状病毒质粒转染于Sf9细胞中;运用间接免疫荧光试验(IFA)、SDS-PAGE和Western blotting试验对Sf9细胞中V5标记Cap蛋白的表达进行验证。结果显示,本试验成功将V5标签引入到PCV2 ORF2基因末端,pFastBacHTAORF2-V5重组转移载体转化大肠杆菌DH10Bac感受态细胞后获得了Bacmid-ORF2-V5重组杆状病毒质粒;IFA、SDS-PAGE和Western blotting试验结果显示,在Sf9细胞中能检测到重组杆状病毒V5标记的PCV2Cap蛋白,具有良好的反应原性,说明本试验成功获得一株rAcMNPV Cap-V5重组杆状病毒。试验结果为PCV2标记亚单位疫苗的研制提供一定的理论依据。     

VP22短肽融合猪圆环病毒2型重组病毒样颗粒的构建及其鉴定

采用融合PCR方法将猪圆环病毒2型(PCV2)Cap基因和人单纯疱疹病毒Ⅰ型病毒(HSV-1)VP22蛋白转导域串联得到Cap-VP22基因,将其插入到杆状病毒转移载体pFastBac Dual构建pFast-Cap-VP22重组转移载体,通过转化含有Bacmid的DH10Bac感受态细胞,经3种抗性和蓝白斑筛选,获得含Cap-VP22基因的重组穿梭质粒rBac-2Cap-VP22。重组穿梭质粒转染昆虫细胞(Sf9),获得重组杆状病毒。重组杆状病毒感染Sf9细胞,通过PCR、间接免疫荧光(IFA)、电镜观察对表达产物进行检测。结果表明,成功构建含双拷贝Cap-VP22基因的杆状病毒载体,IFA证实重组蛋白在Sf9细胞中获得正确表达,与PCV2阳性血清具有良好的反应原性;电镜观察结果显示细胞上清中的目的蛋白可装配形成直径约17nm的病毒样颗粒(VLPs);表明C端融合VP22蛋白转导域序列并不影响Cap蛋白的组装。本研究为进一步研制安全、高效的PCV2疫苗奠定基础。     

猪圆环病毒2型DY株ORF2基因在Sf9昆虫细胞中的表达

本试验采用PCR方法扩增出完整的PCV2 ORF2基因,并将其克隆到Bac-to-Bac杆状病毒表达系统的转移载体pFastBac1^TM中,获得重组转移载体pFast-ORF2,再将其转化DH10Bac感受态细胞,发生转座反应,通过抗性及蓝白斑筛选获得含有ORF2基因的重组杆粒,通过Cellfectin转染试剂介导将重组杆粒DNA转染Sf9昆虫细胞,获得重组杆状病毒。用该重组杆状病毒感染Sf9昆虫细胞,并通过间接免疫荧光法和Western blotting检测目的蛋白的表达。结果表明,ORF2基因在昆虫细胞中获得表达,表达的蛋白质可被PCV2阳性血清识别,这为进一步研究PCV2亚单位疫苗及诊断抗原奠定了基础。     

猪圆环病毒3型Cap蛋白的表达与鉴定

为了表达猪圆环病毒3型(porcine circovirus type 3,PCV3) Cap蛋白,按照昆虫细胞密码子偏嗜性对ORF2基因进行优化并在N端加上His标签基因,合成的ORF2基因片段克隆入pFastBac Dual杆状病毒穿梭载体;将重组质粒转化大肠杆菌DH10-Bac感受态后通过蓝白斑筛选获得重组杆粒Bacmid-C2;将重组杆粒转染至SF9细胞5 d后收集细胞上清液;连续传代3次观察细胞病变。将接毒后的SF9细胞样通过Western blot及间接免疫荧光(IFA)进行Cap蛋白表达的鉴定。利用蔗糖密度梯度离心对病毒样颗粒的Cap蛋白纯化后进行透射电镜观察。结果显示：SDS-PAGE和Western blot检测发现重组杆状病毒在昆虫细胞中能够有效表达Cap蛋白,且与His-tag单抗及PCV3阳性血清发生特异性反应;IFA进一步鉴定感染了重组病毒的昆虫细胞与His单抗产生特异性荧光;透射电镜可以明显观察到14～17 nm左右形态规则的病毒样颗粒(VLPs)。PCV3 Cap蛋白的成功表达和鉴定将为研究Cap蛋白的免疫原性和空间结构奠定基础,也为探讨开发PCV3的亚单...     

PCV3 Cap基因在昆虫杆状病毒中的表达与鉴定

试验旨在获得具有天然构象且反应原性良好的猪圆环病毒3型(porcine circoviurs 3,PCV3)Cap蛋白。以本实验室保存的PCV3 Cap基因为模板,设计不同引物扩增Cap基因,将其亚克隆至pEASY-Blunt载体上,验证正确后将目的基因分别克隆至杆状病毒表达载体pFastBac~(TM)上(含有双启动子),获得含有2个PCV3 Cap基因的重组质粒pFBD-P2C。将该重组质粒转化大肠杆菌DH10-Bac~(TM)感受态细胞当中,进行蓝白斑筛选,获得重组杆状质粒Bacmid P2C,利用脂质体转染至SF9细胞中进行病毒拯救,转染后72 h,收取上清病毒液,盲传3代,收取SF9细胞应用间接免疫荧光法(IFA)及Western blot检测目的蛋白是否表达。结果显示,重组质粒pFBD-P2C酶切验证正确,重组杆粒Bacmid P2C构建成功,杆状病毒中能够表达PCV3 Cap蛋白且能与His-tag抗体发生特异性反应,IFA鉴定出现特异性绿色荧光,表明成功表达了PCV3 Cap蛋白且具有反应原性,为深入开展PCV3抗体制备、新型疫苗研发奠定了基础。     

猪脑心肌炎病毒非结构蛋白3AB基因在重组杆状病毒中的表达与鉴定

利用PCR方法扩增获得EMCV非结构蛋白3AB基因,并将其克隆至杆状病毒转座载体pFastBacTM中,提取阳性克隆质粒转化至DH10Bac感受态细菌,通过蓝白斑筛选和PCR鉴定,得到重组杆状病毒穿梭载体Bacm id-3AB,经脂质体介导转染sf9细胞获得重组杆状病毒,并IFA和W estern b lotting鉴定其抗原性。结果表明,EMCV 3AB基因在昆虫细胞中获得成功表达,分子量约16 ku,具有良好的EMCV抗原性。因此利用杆状病毒表达系统成功表达了EMCV 3AB基因,为该病毒抗原表位和诊断方法研究奠定了重要基础。     

PRRSV变异株ORF6基因重组杆状病毒的构建及其在昆虫细胞的表达

参照GenBank中登录的猪繁殖与呼吸综合征病毒(PRRSV)XH-GD株ORF6(M蛋白)基因序列,设计合成了一对引物,扩增PRRSV ORF6基因.将该基因克隆于转移质粒载体pFcDNA3.1(+)中,获得重组转移质粒pFcDNA3.1-ORF6并将其转化DH10Bac细胞,得到重组穿梭质粒Bacmid-ORF6,再将其转染昆虫细胞Sf9,获得重组杆状病毒rBacmid-ORF6,经PCR鉴定证实目的基因正确地插入到杆状病毒基因组的CMV启动子下游;经过问接免疫荧光试验(IFA)检测,M蛋白在SD细胞中得到了表达,而且表达的产物具有特异免疫学反应性.     

非洲猪瘟P72基因在Bac to Bac系统中的表达及抗原性鉴定

本研究旨在通过在Bac to Bac系统中表达非洲猪瘟病毒(ASFV)的P72基因,为进一步研究P72基因的结构、功能和免疫学特性奠定基础。本研究从含P72全长基因的菌株pGEX-6p-1-P72中扩增出P72基因全序列1941 bp,将扩增的基因连接于pFastBac HTa杆状病毒载体中,构建重组质粒pFastBac HTa-P72,转化至感受态DH10Bac中,获得重组杆粒rBacmid-P72,再将其转染至Sf9昆虫细胞,获得重组杆状病毒。经Western blotting和夹心ELISA分析,结果表明,P72在昆虫细胞Sf9中获得正确表达,重组P72蛋白可被特异性兔抗ASFV P72血清、P72单抗识别。结果提示,Bac to Bac系统表达的P72重组蛋白特异性强、活性稳定,具有良好的抗原性,为快速诊断ASF提供良好的候选抗原。     

表达猪圆环病毒2型Rep蛋白的重组杆状病毒的构建与鉴定

猪圆环病毒2型(PCV2)是近年来新发现的引起断奶仔猪多系统衰弱综合征的主要病原,含有两个主要的阅读框ORF1和ORF2,分别编码复制相关蛋白(Rep)和核衣壳蛋白(Cap)。为了探究ORF1基因结构及其与功能的关系,本研究利用Bac-to-Bac杆状病毒表达系统将PCV2的ORF1基因克隆到杆状病毒转移载体pFastBacTMDual中,获得重组转移载体pFBD-ORF1,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ORF1基因的重组穿梭载体rBacmid-ORF1,经脂质体介导转染昆虫细胞sf9,获得重组杆状病毒rAc-Rep,SDS-PAGE和Western-blotting分析可见大小约为36ku的特异性条带,表明rAc-Rep在sf9中成功表达了PCV2Rep蛋白。     

H9亚型禽流感病毒基质蛋白M1单克隆抗体的制备与鉴定

本研究用纯化的H9N2亚型禽流感病毒免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合。对杂交瘤细胞及时筛选,阳性孔经3次有限稀释法克隆,成功获得3株能稳定传代并分泌抗H9亚型禽流感病毒基质蛋白M1单克隆抗体的杂交瘤细胞：3G8、2F6、5F2。间接ELISA方法检测,3株单克隆抗体的腹水间接ELISA效价达106以上。构建了真核表达载体pCAGGS-M1并转染于MDCK细胞,以用于腹水的Western blot和间接免疫荧光鉴定。间接免疫荧光结果表明,3株单克隆抗体皆与真核表达蛋白M1反应。这些单克隆抗体的制备为后期研究M1蛋白在流感病毒复制与出芽过程中的重要生物学功能奠定了基础。     

Preparation of Monoclonal Antibodies against Porcine Circovirus Type 2 Cap Protein

The structural protein Cap encoded by ORF2 of porcine circovirus type 2 (PCV2) was expressed in genetic engineering recombinant bacteria and used as the immunogen after purification.Five hybridoma cell lines against PCV2 Cap protein named as 3D12,4D5,4B9,4C9 and 4G10,respectively,were developed after fusion between SP2/0 myeloma cells and spleen cells of BALB/c mice immunized with purified recombinant PCV2 Cap protein.Except the heavy chain type of 4D5 was identified as IgG2b,others were identified as IgG1;The light chains were all kappa.In Western blotting assay,all the monoclonal antibodies (mAbs) couldn't specifically recognize the 47 ku recombinant PCV2 Cap protein,but showed strong specific fluorescence in PCV2 infected PK15 cells in IFA,which indicated that all the mAbs recognized comformational epitope.The neutralization test showed that all the mAbs had neutralization activity.These results laid the foundation for further study of the structure and function of PCV2 ORF2 gene,and establishment of the method for diagnosing PCV2 rapidly and exactly.     

猪圆环病毒2型Cap蛋白单克隆抗体的制备

本研究利用已构建的基因工程重组菌表达猪圆环病毒2型(porcine circovirus type 2,PCV2)ORF2编码的Cap蛋白,纯化后作为免疫原,免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞融合。经间接免疫荧光试验(IFA)筛选及有限稀释法进行3次亚克隆后,本试验最终获得5株稳定分泌抗PCV2 Cap蛋白单克隆抗体的杂交瘤细胞,分别命名为3D12、4D5、4B9、4C9和4G10。其中4D5为IgG2b亚型,其余4株单克隆抗体均为IgG1亚型,轻链类型均为κ。Western blotting鉴定结果表明获得的5株单克隆抗体均不能特异性的识别47 ku重组PCV2 Cap蛋白;对病毒感染细胞进行IFA试验,结果显示5株单克隆抗体均特异性的识别病毒抗原,表明5株单克隆抗体识别的抗原表位均为构象表位。中和试验结果表明,5株单克隆抗体均有中和活性。本试验结果为进一步探索ORF2基因的结构、功能及建立快速准确的诊断方法奠定了基础。     

Production and characterization of monoclonal antibodies against porcine interleukin-4

Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. In the present study, recombinant thioredoxin (Trx)-porcine IL-4 (pIL-4) fusion protein was prepared by Escherichia coli (E. coli), and by using this protein as an immunogen, monoclonal antibodies (mAbs) against pIL-4 were produced to establish a basis for a research on immune responses in pigs. Six stable hybridoma cell lines were successfully established and specific binding of each mAb to recombinant pIL-4 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that the subclass of 5 out of 6 mAbs was IgG1 and the rest was IgG2b. Further, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to 4 different epitopes. The recombinant proteins and mAbs produced in this study will be useful tools for the assessment of porcine immune system.     

猪圆环病毒2型ORF2和猪繁殖与呼吸综合征病毒ORF5重组杆状病毒疫苗的研究

参照GenBank上收录的基因组序列自行设计引物,分别扩增猪圆环病毒2型ORF2和猪繁殖与呼吸综合征病毒ORF5。将外源基因分别插入经改造的杆状病毒穿梭载体pFVC,获得3种重组质粒pFVC-ORF2、pFVC-ORF5、pFVC-ORF5-ORF2。将重组质粒pFVC-ORF2、pFVC-ORF5、pFVC-ORF5-ORF2分别转入带有杆状病毒骨架的DH10BacTME.coli感受态细胞中。提取高质量的重组质粒rBacmid-ORF2、rBacmid-ORF5、rBacmid-ORF5-ORF2,将重组质粒转染Sf9细胞,获取重组病毒rAc-ORF2、rAc-ORF5、rAc-ORF5-ORF2。通过间接免疫荧光检测证实了外源基因正确表达。重组杆状病毒rAc-ORF2、rAc-ORF5、rAc-ORF5-ORF2对BALB/c小鼠的免疫试验结果显示,rAc-ORF2、rAc-ORF5、rAc-ORF5-ORF2均能够刺激小鼠机体产生免疫应答,产生抗体,并能持续较长时间。     

Development of a monoclonal antibody-based sandwich ELISA for detection of guinea pig interleukin-2

Interleukin 2 (IL-2) is a T cell proliferation factor released by Th0- and Th1-type helper T cells and is an essential cytokine for immune responses. In the present study, recombinant glutathione S-transferase (GST)-guinea pig IL-2 (GPIL-2) fusion protein was prepared by Escherichia coli (E. coli) and by using this protein as an immunogen, monoclonal antibodies (mAbs) against GPIL-2 were produced to establish a basis for a research on immune responses in guinea pigs. Three stable hybridoma cell lines were established, and specific binding of each mAb to recombinant GPIL-2 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that all three mAbs were IgG1 and had kappa chain. Furthermore, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to three different epitopes. Thus, a sandwich ELISA based on the two mAbs specific to different GPIL-2 epitopes was developed for detection of GPIL-2, which had a sensitivity threshold of about 0.3 ng/ml of GPIL-2.     

传染性造血器官坏死病病毒糖蛋白杆状病毒表达系统的构建及其抗原性分析

为研究传染性造血器官坏死病病毒（infectious haematopoietic necrosis virus,IHNV）主要结构蛋白糖蛋白（G）,本研究采用RT-PCR方法从IHNV中提取RNA并进行反转录,经PCR方法扩增获得1 380 bp的G蛋白基因片段,将其克隆到pFB-LIC-Bse杆状病毒载体中,成功构建了重组质粒pFB-LIC-Bse-G,转化到大肠杆菌DH10Bac感受态细胞中,获得了重组杆粒rBacmid-G。将重组杆粒转染至Sf9昆虫细胞,获得了重组杆状病毒。间接免疫荧光（IFA）和Western blotting分析显示,重组G蛋白可与抗组氨酸单抗（Anti-His）、抗IHNV鼠血清发生特异性反应,表明本研究克隆的IHNV G蛋白在真核表达系统中得到正确表达,且该蛋白具有良好的抗原性。本试验结果为研究G蛋白的功能及开发IHNV新型疫苗奠定了基础。     

Construction of Baculovirus Expression System and Antigenicity Analysis of Infectious Hematopoietic Necrosis Virus Glycoprotein

To study the major structural protein glycoprotein (G) of infectious hematopoietic necrosis virus (IHNV), glycoprotein gene (1 380 bp) was amplified by RT-PCR from IHNV. In order to construct a recombinant plasmid pFB-LIC-Bse-G, G gene was cloned into the baculovirus vector pFB-LIC-Bse. Then, the constructed plasmid pFB-LIC-Bse-G was transformed into E.coli DH10Bac. The recombinant bacmids rBacmid-G was got, and then it was transfected to insect Sf9 cells,and the recombinant baculovirus that contained G gene was obtained. Western blotting analysis and indirect immunofluorescence assay (IFA) showed that the recombinant G protein could be recognized by histidine monoclonal antibody (Anti-His) and anti-IHNV antibody of mouse. The results indicated IHNV G protein had been expressed correctly in Sf9 cells. The study laid a foundation for further studying the G protein structure, function and immunological characteristics.