猪戊型肝炎病毒ORF3基因的克隆与原核表达

为了表达猪源戊型肝炎病毒衣壳蛋白,并对其反应原性进行研究,采用RT-PCR方法扩增猪戊型肝炎病毒(SHEV)ORF3完整基因,将扩增产物插入pMD19-T载体,再亚克隆至原核表达载体pET-32a(+),构建了pET-32a(+)-ORF3表达载体,转入E.coli BL21(DE3),IPTG诱导表达,同时对诱导表达条件进行筛选,并用SDS-PAGE和免疫印迹等方法分析鉴定表达产物。结果表明,成功扩增到345bp的目的基因片段,表达载体pET-32a(+)-ORF3转入E.coli BL21(DE3),经IPTG诱导,成功表达了ORF3基因编码的蛋白,当IPTG浓度为1mmol/L,诱导时间为6h时,该基因在E.coli BL21(DE3)中表达量最高。表达产物的分子质量约为29.6ku,与预期目的蛋白分子质量相符。表达的蛋白既有包涵体形式,也有可溶性形式。经Western blot检测,表达的目的蛋白能与SHEV阳性血清发生特异性反应。     

猪瘟病毒多表位基因的表达及其免疫原性分析

体外表达猪瘟病毒(CFSV)T、B细胞表位,并对表达产物的免疫原性进行分析.人工合成CFSV的多个T、B细胞表位及表位之间的连接linker基因,并将该基因插入pGEX-6P-1表达载体,经酶切和测序鉴定获得重组阳性克隆,成功构建了CFSV复合多表位抗原基因的原核表达质粒pGEX-BT500,转化E.coli BL21,IPTG诱导表达,纯化表达产物并免疫兔.结果:通过IPTG诱导目的基因可高效表达,SDS-PAGE结果表明,以终浓度为0.9 mmol· L-1的IPTG进行诱导,7h后表达量最高,产物分泌表达,相对分子质量约43 ku,表达产量约占菌体总蛋白的30％.Western blotting检测表明,表达的融合蛋白能与猪瘟阳性血清发生特异性反应;兔攻毒试验表明所免疫表达产物可保护(根据发热反应评价)兔.结果表明表达获得的产物具有良好的反应原性,这为应用该融合蛋白制备CSFV免疫血清学诊断试剂和多表位疫苗研究奠定了基础.     

微小隐孢子虫E2F样结构域包含转录因子CpELD基因的原核和真核表达

为了实现微小隐孢子虫(Cryptosporidium parvum)E2F样结构域包含转录因子编码基因cpeld的原核和真核表达,设计2对特异性引物,以C.parvum卵囊cDNA为模板,PCR扩增得到cpeld基因,将其分别插入pET32a和p3XFLAG-CMV14载体。同时设计引物,扩增微小隐孢子虫类钙调蛋白(C.parvum calmoduin-like proteins)的编码基因CpCML,插入pcmv-HA载体。将鉴定正确的重组质粒分别转入BL21(DE3)和293T细胞进行表达,Western blot观察重组蛋白的反应原性。结果显示,成功构建了重组原核表达质粒pET32a-cpeld和真核表达质粒p3XFLAG-CMV14-cpeld以及pcmv-HA-CML;SDS-PAGE显示,重组质粒转化菌经IPTG诱导后表达出了分子质量约为55 ku的重组蛋白;Western blot分析显示,原核表达的重组蛋白能与鼠抗His单克隆抗体,真核表达重组CpELD和CpCML蛋白能分别与抗FLAG和HA单克隆抗体反应。结果表明,成功实现了cpeld基因的原核和真核表达以及CpCML基因的真核表达,表达产物具有良好的反应原性,为后续开展隐孢子虫CpELD和CpCML的功能和作用机制研究、新型的防控技术研发奠定了基础。     

旋毛虫T3223-6 cDNA的表达及鉴定

利用PCR技术将T3223-6 cDNA扩增克隆到原核表达载体pET-28a,将重组质粒转入克隆菌Nova-blue,提取质粒进行酶切和测序鉴定后转入表达菌BL21star(DE3).用1 mM IPTG诱导培养重组表达菌,对菌体裂解物进行SDS-PAGE分析,检测重组蛋白的表达情况.用旋毛虫感染猪血清和正常血清,通过western blotting检测重组蛋白的反应原性.结果表明:经IPTG诱导后重组转化菌的裂解产物出现44 kD左右的表达带,大小与理论值相符;western blotting检测结果显示重组蛋白可以被旋毛虫感染猪血清识别,具有反应原性.     

猪流行性腹泻病毒COE蛋白原核表达及其反应原性的初步鉴定

为了研究猪流行性腹泻病毒(PEDV)的优势抗原表位COE可溶性原核表达及其反应原性,试验采用RT-PCR方法扩增出COE基因,将其克隆至原核表达载体p MAL-C5X上,构建重组质粒p MAL-C5X-COE,经测序鉴定正确后转化Rosetta感受态细胞,并进行IPTG诱导表达和Westernblot验证。结果表明:重组菌p MAL-C5X-COE表达的融合蛋白MBP-COE的分子质量约为57 ku,优化的诱导条件为IPTG浓度0.8 mmol/L、诱导时间4 h,诱导温度30℃,重组蛋白以可溶性蛋白形式存在于菌体中,融合蛋白与兔抗PEDV多克隆抗血清发生特异的免疫印迹反应。说明原核表达的COE融合蛋白可溶且具有良好的反应原性。     

猪蛔虫rAs37基因的克隆及原核表达

根据编码rAs37的已知基因序列设计并合成1对引物,应用PCR技术从猪蛔虫感染性虫卵的cDNA中扩增编码rAs37的部分基因,克隆入pGEX-KG表达载体,转化入E.coli DH5α感受态细胞,经含氨苄青霉素琼脂平板筛选,小量抽提质粒进行酶切、PCR及DNA测序鉴定.然后阳性重组质粒转化入E.coli BL21-CodonPlus, IPTG诱导,表达产物经SDS-PAGE和Western-blot分析鉴定.结果显示,扩增的rAs37基因与GenBank中相应基因序列(AB078971)的同源性达100%,表达的rAs37融合蛋白表观相对分子质量约为60 000,且可被兔抗猪蛔虫免疫血清识别.说明所获得的表达蛋白质具有一定的反应原性,为下一步利用重组蛋白建立猪蛔虫早期诊断方法和研制猪蛔虫的亚单位疫苗奠定了基础.     

弓形虫微线体蛋白MIC3基因的克隆及原核表达

根据编码MIC3的已知基因序列设计并合成一对引物,应用PCR技术从弓形虫RH株的基因组DNA中扩增编码MIC3的全长基因,克隆入pGEX-KG表达载体,转化入E.coli DH5α感受态细胞,经含氨苄青霉素琼脂平板筛选,小量抽提质粒进行酶切、PCR及DNA测序鉴定.然后阳性重组质粒转化入E.coli BL21-CodonPlus,IPTG诱导,表达产物经SDS-PAGE和Western-blot分析鉴定.结果显示,扩增的MIC3基因与GenBank中相应基因序列（AJ132530）的同源性达99.6 %,表达的MIC3融合蛋白表观分子量约为66 ku,且可被兔抗弓形虫免疫血清识别.说明所获得的表达蛋白质具有一定的反应原性,为下一步利用重组蛋白建立弓形虫的诊断方法和研制弓形虫的亚单位疫苗奠定了基础.     

西尼罗病毒糖蛋白第三结构域的原核表达及鉴定

根据西尼罗病毒(West Nile virus,WNV)序列设计1对特异性引物,用PCR反应扩增糖蛋白第三结构域(WNVEDⅢ)基因,将PCR产物克隆至pGEX-4T-1原核表达载体上,获得重组表达质粒,将该质粒转化入E.coli DH5α,命名为pG-EDⅢ,鉴定正确后转入transetta表达菌中,经IPTG诱导后纯化重组蛋白,通过SDS-PAGE和Western-blot方法证实WNV-EDⅢ基因可在大肠杆菌中高效表达,小鼠免疫试验证明该原核表达蛋白有良好的免疫原性。     

犬细小病毒NY株VP2蛋白的原核表达及反应原性鉴定

犬细小病毒(CPV)编码的VP2基因容易发生变异,导致病毒嗜性和致病性的改变。为制备一株高致病性的CPV毒株(NY株,基因型为2a型)重组VP2蛋白,构建重组原核表达载体pET28a-CPVVP2,转化E.coli BL21(DE3)表达菌株,通过不同温度、不同IPTG浓度和不同诱导时间等条件的优化进行表达蛋白,采用SDS-PAGE和Western blot进行蛋白鉴定。结果表明,重组CPV-NY毒株VP2蛋白(rVP2)的分子质量约为72ku,在不同温度条件下均以包涵体形式存在,在37℃条件下以终浓度为0.2mmoL/L IPTG诱导4h表达量达到最高。rVP2不仅与His标签mAb反应,也能与CPV特异性阳性血清反应,说明rVP2具有良好的反应原性,为CPV抗体检测试剂盒和基因工程疫苗的研究奠定了基础。     

环形泰勒虫表面抗原重组蛋白Tasp-Tams1-Spag1生物信息学分析与原核表达

运用生物信息学软件对环形泰勒虫表面抗原串联基因Tasp-Tams1-Spag1进行分析,预测其编码蛋白的主要特性与抗原表位等,并对此串联基因进行克隆与原核表达。结果表明:串联重组蛋白Tasp-Tams1-Spag1属于不稳定非分泌型亲水性蛋白;编码346个氨基酸;有4个低复杂性的结构域,且含有Sorb与Btz、NL、NOT的同源区域;可能存在11个B细胞表位优势区段与17个T细胞表位优势区段,含有T、B细胞联合表位,表明Tasp-Tams1-Spag1重组蛋白可能具有良好的免疫原性。IPTG诱导重组蛋白原核表达,结果显示,该重组蛋白以包涵体形式存在;经Western blot检测,表明此串联蛋白反应原性良好,为后续深入研究免疫抗原奠定基础。     

Antigen heterogeneity and epitope variable expression in Mycoplasma meleagridis isolates

Immunoblotting was used to check the antigenic profiling of 27 Mycoplasma meleagridis strains isolated in different countries. Hyperimmune polyclonal rabbit antiserum as well as monoclonal antibodies (MAbs) raised against M. meleagridis (MM) showed antigen heterogeneity among strains. Five anti-MM MAbs were selected for lack of reaction against heterologous avian mycoplasma. Three of these five Mabs did not cross-react with 63 mycoplasma strains from six species affecting turkeys other than M. meleagridis. The five Mabs used to analyse the epitopes of 30 M. meleagridis strains indicated that some epitopes were not expressed in all strains. Moreover, other epitopes were located on proteins which differed according to number or molecular mass from strain to strain. The five Mabs therefore, recognised variable surface proteins, among which two were amphiphilic membrane proteins.Three of the selected Mabs recognised 29 or 30 of the 30 tested strains. The in vitro expression of surface epitopes in M. meleagridis ATCC 25284 was investigated by colony immunobinding and allowed demonstration of a variable antigenic system.     

Preparation and partial characterization of monoclonal antibodies against the protective protein antigen of Erysipelothrix rhusiopathiae

Thirteen mouse monoclonal antibodies (Mabs) against the protective protein antigen (P64) of Erysipelothrix rhusiopathiae were prepared and partially characterized. The titres of the Mabs varied from 200 to 1,638,400 as determined by enzyme-linked immunosorbent assay (ELISA). Of the 13 Mabs 10, two and one belonged to the IgG2a, IgG1 and IgM subclasses, respectively. All Mabs reacted strongly with the 64 kDa protein and weakly with the 43 kDa protein upon Western blotting of the alkaline extract (AE) of E. rhusiopathiae. The protective activity (PD50/ml) of the 13 Mabs against E. rhusiopathiae infection in mice varied from < 50 to > 50,000. These Mabs were classified into three groups, highly protective Mabs, moderately protective Mabs and Mabs which did not possess protective activity, based on the protective index (ratio of the PD50/ml to the antibody titre). These results suggest that the 64 kDa protein is an effective protective antigen, which is easily cleaved into many small proteins, including the 43 kDa protein, and possesses at least two epitopes related to its protective activity and at least one epitope which is not related to protection of mice against E. rhusiopathiae infection.     

Monoclonal antibody against bovine alpha1-acid glycoprotein

Monoclonal antibodies (Mabs) against bovine alpha1-acid glycoprotein (alpha1AGP) were prepared from mouse hybridoma cell line. Bovine alpha1AGP as antigen was purified by using ion exchange column chromatography and the yield from 500 ml serum was about 10 mg. Immunoglobulin isotypes of 3 Mabs obtained were IgM and light chain types were kappa. The Mabs reacted with bovine alpha1AGP on immunoblot analysis, but not with alpha1AGP digested with N-glycosidase, suggesting that an epitope recognized by these Mabs may be associated with a glycan side chain of bovine alpha1AGP.     

猪戊型肝炎病毒Ⅳ型衣壳蛋白单克隆抗体的制备与鉴定

为获得猪戊型肝炎病毒(Hepatitis E virus,HEV)Ⅳ型衣壳蛋白单克隆抗体,将猪HEV衣壳蛋白的C端267(408—675)个氨基酸基因序列克隆入原核表达载体pET-28a(+),构建重组质粒pET-28a-ORF2-C,转化E.coli Rosetta(BL21)感受态细胞进行诱导表达,SDS-PAGE和Western blot鉴定,纯化后免疫小鼠。取免疫小鼠的脾脏与鼠骨髓瘤细胞SP2/0融合制备单克隆抗体。通过间接ELISA和竞争ELISA方法筛选并鉴定单抗。结果表明蛋白得到正确、高效表达,获得3株识别不同的抗原表位区的单克隆抗体,分别命名为Mab-1E4(IgG1)、Mab-2C7(IgG1)和Mab-2G9(IgG2b),其中1E4和2G9能阻断临床阳性猪血清,提示该2株单克隆抗体识别的抗原表位是猪HEVⅣ型衣壳蛋白上重要的抗原表位区,而单抗Mab-2C7不能阻断。本研究为猪HEVⅣ型的诊断及研究提供重要工具。     

Production and characterization of monoclonal antibodies to bovid herpesvirus-4

Thirty-five hybridoma cell lines secreting monoclonal antibodies (Mabs) against bovid herpesvirus-4 (BHV-4) strain V. Test were produced. These hybrid cells resulted from the fusion of SP2/0 myeloma cells with splenocytes of BALB/c mice previously immunized with purified BHV-4. A modified indirect fluorescent antibody test (IFAT) was applied as a screening procedure and was compared with an indirect enzyme-linked immunosorbent assay. The selected Mabs were tested by the same IFAT against a panel of BHV-4 field isolates and against bovine herpesvirus-1, bovine herpesvirus-2 and alcelaphine herpesvirus-1 (AHV-1). Comparison of BHV-4 field isolates with Mabs confirmed their close antigenic relationships, but slight antigenic differences were observed between different isolates. One of the Mabs also reacted against AHV-1, indicating an antigenic relationship between BHV-4 and AHV-1. None of the Mabs reacting with BHV-4 possessed neutralizing activity against the strain used for immunization.     

1株非洲猪瘟病毒p30蛋白单克隆抗体识别的B细胞抗原表位的鉴定

旨在研究非洲猪瘟病毒(ASFV) p30蛋白的B细胞抗原表位,本研究制备了p30蛋白的单克隆抗体(mAb),并以该单克隆抗体为工具进行B细胞抗原表位定位。首先,通过原核表达及Ni柱亲和纯化获得p30蛋白,将纯化蛋白免疫BALB/c小鼠进行杂交瘤细胞制备,通过间接酶联免疫吸附试验(iELISA)筛选出阳性杂交瘤细胞,并以细胞表达的方法制备单克隆抗体;采用间接免疫荧光试验(IFA)和蛋白质免疫印迹(Western blot)对单克隆抗体的特异性进行鉴定。利用IEDB表位预测软件对p30蛋白B细胞抗原表位进行预测,根据预测结果对CP204L基因进行截短表达,利用IFA、Western blot和iELISA对其抗原表位进行鉴定。最后,利用噬菌体十二肽库对制备的p30单克隆抗体进行4轮生物淘选,筛选多肽表位,并与上述基因截短表达筛选方法进行比对。特异性鉴定结果显示,该单克隆抗体能成功识别感染猪肺泡巨噬细胞的ASFV;基因截短表达筛选结果表明,其识别的抗原表位区域为⁸⁴M～K¹⁴²;噬菌体淘选试验结果表明,¹¹⁶TSSFETLFE¹²⁴为本试验制备的单克隆抗体所识别的p30蛋白抗原表位核心序列,该结果进一步缩小了表位分布范围。本研究制备了p30蛋白的1株单克隆抗体,并对其抗原表位进行鉴定,为血清学诊断试剂的研发和p30蛋白功能研究奠定基础。     

Identification of Actinobacillus pleuropneumoniae strains of serotypes 7 and 4 using monoclonal antibodies: demonstration of common LPS O-chain epitopes with Actinobacillus lignieresii

Monoclonal antibodies (Mabs) against Actinobacillus pleuropneumoniae serotype 7 were produced and characterized. Three Mabs directed against surface polysaccharides were selected. One of the Mabs was directed against a capsular polysaccharide epitope (CPS) of A. pleuropneumoniae serotype 7 whereas two other Mabs reacted with different epitopes of the LPS O-chain. One of the latter reacted with the reference strain of serotype 7 and the other one with serotypes 7 and 4. These three Mabs were used to test, by Dot-ELISA, 508 field strains of A. pleuropneumoniae. None of the strains belonging to other serotypes different from serotypes 4 and 7 were positive with the Mabs. Used in combination, the CPS and one of the LPS O-chain directed Mabs were shown to be suitable for serotyping since they detected 100% of serotype 7 strains. In this study, we confirm for the first time that A. pleuropneumoniae serotype 4 is present in North America. Finally, both O-chain specific Mabs also reacted with the O-chain of Actinobacillus lignieresii. The cross-reactivity between the two species was confirmed using sera from pigs experimentally infected with A. pleuropneumoniae serotype 7 and A. lignieresii, using immunoblotting and ELISA. This is the first report of a specific cross-reactivity between the LPS of these bacterial species.     

The ovine cytotoxic T lymphocyte responses to bluetongue virus

This study uses recombinant vaccinia viruses expressing truncated or entire bluetongue virus (BTV) proteins to map the location of epitopes recognized by cytotoxic T lymphocytes (CTL) from Australian merino sheep. The non-structural protein, NS1, was recognised by CTL from all sheep, while VP2, VP3, VP5 and VP7 were recognised by CTL from only some sheep. The remaining proteins (except for VP1, which was not tested) did not contain CTL epitopes. When truncated genes were used to map the location of CTL epitopes, it was found that sheep often have CTL that recognise more than one epitope in NS1 or VP2. Overall there was considerable diversity in the CTL recognition patterns in the sheep tested.     

Characterisation of monoclonal antibodies to Epizootic Hemorrhagic Disease Virus of deer ( ) and Bluetongue Virus by immunisation of mice with recombinant VP7 antigen

Immunisation of mice with recombinant VP7 antigen of epizootic hemorrhagic disease virus of deer ( ) induced serum antibody responses to . However, from the 19 monoclonal antibodies (Mab) produced from these mice, 15 were specific for and four for bluetongue virus ( ). No Mabs were identified with the specificity for an epitope of VP7 shared by both and in spite of the fact that they share a large portion of homology in VP7 amino acids composition. These Mabs were divided into five groups based on their specificity and interaction with each other. Group II Mabs, consisting of 13 Mabs, recognises a potential serogroup specific, linear epitope of VP7 antigen. One of the Mabs to (Group V) was identified as VP7 specific with the possibility of being the serogroup specific and recognizes a potential conformational epitope. Two Mabs from these VP7 specific groups were further analysed and found to be useful in a competitive enzyme-linked immunosorbent assay ( - ) for detection of specific antibodies against and in bovine sera.     

Characterisation of monoclonal antibodies to epizootic hemorrhagic disease virus of deer (EHDV) and bluetongue virus by immunisation of mice with EHDV recombinant VP7 antigen.

Immunisation of mice with recombinant VP7 antigen of epizootic hemorrhagic disease virus of deer (EHDV) induced serum antibody responses to EHDV. However, from the 19 monoclonal antibodies (Mab) produced from these mice, 15 were specific for EHDV and four for bluetongue virus (BTV). No Mabs were identified with the specificity for an epitope of VP7 shared by both EHDV and BTV in spite of the fact that they share a large portion of homology in VP7 amino acids composition. These Mabs were divided into five groups based on their specificity and interaction with each other. Group II Mabs, consisting of 13 Mabs, recognises a potential serogroup specific, linear epitope of EHDV VP7 antigen. One of the Mabs to BTV (Group V) was identified as BTV VP7 specific with the possibility of being the serogroup specific and recognizes a potential conformational epitope. Two Mabs from these VP7 specific groups were further analysed and found to be useful in a competitive enzyme-linked immunosorbent assay (C - ELISA) for detection of specific antibodies against EHDV and BTV in bovine sera.