Cloning and characterization of cDNA encoding a prohibitin-like protein from Theileria orientalis

A cDNA clone encoding a prohibitin-like protein (Toprh) was isolated from a piroplasm cDNA library of Theileria orientalis and its nucleotide sequence was determined. An open reading frame, encoding a polypeptide of 278 amino acid residues, was found in Toprh cDNA sequence. An intron of 89 bp was identified when this cDNA clone was compared with the Toprh gene in the genome of T. orientalis. The deduced amino acid sequence of Toprh shares 93.8, 93.1 and 69.1% identities with the prohibitins of T. parva (from chromosome 1), T. annulata (from chromosome 1), and Plasmodium falciparum, (from chromosome 10), respectively. By Western blot analysis, Toprh was found to be expressed in the piroplasm stage of the parasites.     

猪圆环病毒2型CC株ORF3基因的克隆与原核表达

为原核表达猪圆环病毒2型(PCV2)的ORF3编码蛋白,本研究采用PCR方法以PCV2的CC株的基因组DNA为模板扩增ORF3基因,将其克隆到原核表达载体pET-32a(+)中,转化大肠杆菌BL21 (DE3),经IPTG诱导表达.SDS-PAGE结果显示,表达的重组蛋白约为31ku,并且以包涵体形式存在;western blot分析表明,ORF3重组蛋白能够与鼠抗His标签单克隆抗体发生特异性反应,表明原核表达的ORF3重组蛋白具有良好的反应原性.     

牛病毒性腹泻病毒E2蛋白的多克隆抗体制备及鉴定

为制备牛病毒性腹泻病毒(BVDV)重组E2蛋白的兔源多克隆抗体,本研究利用表达BVDV E2蛋白的重组质粒pET30a-E2转化E.coli BL21(DE3),经诱导表达获得重组E2蛋白。Western blot检测显示纯化蛋白能够与BVDV参考阳性血清反应。以纯化的重组E2蛋白免疫新西兰白兔制备多克隆抗体,病毒中和试验测定其中和效价为1:2048,间接免疫荧光和western blot试验表明其具有良好的反应性和特异性。本研究制备的BVDV重组E2蛋白兔源多克隆抗体可应用于BVDV的检测,同时为进一步建立检测BVDV E2蛋白的ELISA方法奠定基础。     

Molecular epidemiological survey of Theileria orientalis in Thua Thien Hue Province, Vietnam

Theileria orientalis is a benign bovine protozoan parasite that occasionally causes serious economic loss in the livestock industry. We report the findings of a molecular epidemiological survey of T. orientalis in 94 Vietnamese yellow cattle, 43 water buffaloes, 21 sheep, 21 goats and 85 blood-sucking ticks of cattle in the Thua Thien Hue province of Vietnam. The major piroplasm surface protein (MPSP) gene of T. orientalis was detected using polymerase chain reaction from 13 cattle (13.8%), 11 water buffaloes (25.6%), 1 sheep (4.8%) and 9 ticks (10.6%). Phylogenetic analysis using MPSP gene sequences showed the presence of seven genotypes, four previously categorized genotypes (Types 1, 3, 5 and 7) and three new genotypes (Types N-1, N-2 and N-3).     

Prokaryotic Expression and Immunogenicity Analysis of Hemagglutinin-related Outer Membrane Protein from F.necrophorum

In order to analyze the immunogenicity of 120 ku hemagglutinin-related outer membrane protein from F.necrophorum,specific primers were designed to amplify the 3 truncated overlapping gene fragments covering the whole open reading frame (ORF) based on the result of antigenic epitope analysis.The gene fragments were ligated into the prokaryotic expression vector to construct pET-28a-p1,pET-32a-p2 and pET-32a-p3,respectively.Then recombinant plasmid was induced expression in E.coli.The SDS-PAGE results showed that the expression of recombinant proteins P1,P2 and P3,molecular mass of the expressed proteins were about 48,59 and 62 ku,respectively.Western blotting indicated that the recombinant proteins could be recognized by antiserum against F.necrophorum from mouse.Humoral immunity response against recombinant proteins was detected in the immunized rabbits.Altogether,these findings suggested that 120 ku hemagglutinin-related outer membrane protein from F.necrophorum had good immunogenicity,and this study provided a reference for further research on genetic engineering subunit vaccine of F.necrophorum.     

牛朊蛋白27-30基因的克隆与表达

利用PCR技术，从含有牛朊蛋白（Prion protein，PrP）基因Prnp的开放阅读框的克隆质粒BoPrnp—T中扩增出约420bp的目的基因（PrP猢基因）。将PrP^27-30基因和载体pPIC9K分别用限制性核酸内切酶EcoRⅠ和NotⅠ进行双酶切，T4DNA连接酶作用后，转化至E．coli JM109中，构建重组表达载体pPIC9K-boPrP^27-30。pPIC9K-boPrP^27-30经限制性核酸内切酶SalⅠ线性化后电转至毕赤酵母GS115中，经G418筛选后得到高拷贝的重组菌株GS115／pPIC9K-boPrP^27-30。GS115／pPIC9K-boPrP^27-30经1．0％甲醇诱导后，表达产物用SDS-PAGE和Western blot分析，结果表明牛PrP^27-30基因在毕赤酵母细胞中获得表达，表达产物的分子量约为27Ku，能够被单克隆抗体SAF-70识别。     

猪圆环病毒2型GD株ORF2基因重组腺病毒的构建及其表达

根据PCV2ORF2基因的序列设计两条引物从PCV2的PK15细胞培养物中扩增出ORF2基因(702 bp).将此基因片段克隆入pMD 18-T载体,经酶切、测序鉴定筛选出重组质粒pMD-ORF2.将PCV2 ORF2基因克隆入pAdeasy腺病毒载体系统的穿梭质粒pAdTrack-CMV中,获得重组穿梭质粒pAdTrack-CMV-ORF2,将重组质粒与腺病毒骨架载体共转化大肠杆菌BJ5183,通过细菌内同源重组获得重组腺病毒质粒pAdCMV-ORF2,然后用PacⅠ线性化后转染293细胞,在293细胞内包装出重组腺病毒.通过荧光显微镜观察、PCR检测和Western blot检测证明PCV2 ORF2基因在293细胞内获得表达,ORF2多肽具有良好的免疫原性.     

牛瑟氏泰勒虫MPSP双表位基因的克隆及原核表达

为了探究表位疫苗是否可以应用于预防牛瑟氏泰勒虫(Theileria sergenti),根据MPSP (Major piroplasm surface protein)基因序列(FJ515689.1)设计合成了2对引物,以T.sergenti基因组DNA为模板,通过SOE-PCR扩增出长约361 bp的双表位基因融合片段,2个表位基因通过linker (Gly4Ser)3相连.将该片段连接pGEX-4T-2,构建pGEX-4T-E1-2重组表达载体,转化BL21,IPTG诱导表达,经SDS-PAGE电泳显示表达了约39 ku的融合蛋白.Westem blot分析表明该双表位重组蛋白可与T.sergenti阳性血清发生特异性反应,表明融合蛋白具有反应原性.     

Genotypic diversity of Theileria orientalis detected from cattle grazing in Kumamoto and Okinawa prefectures of Japan

Theileria orientalis is a benign protozoan species that is widely distributed in Japan, yet sometimes causes serious economic losses in the livestock industry. In this study, we conducted a molecular survey based on genes encoding the major piroplasm surface protein (MPSP) and p23 for T. orientalis detected in cattle grazing in southern areas of Japan, consisting of 2 farms in Kumamoto prefecture (Aso and Kuma districts) and 3 farms in Okinawa prefecture (Ishigaki, Iriomote, and Yonaguni Islands). High prevalence rates of T. orientalis infection were shown in all the cattle populations using the diagnostic MPSP- and p23-PCR assays. Phylogenetic analyses revealed 4 MPSP genotypes and 3 p23 genotypes. Furthermore, MPSP genotype-specific PCR methods were developed in this study and wide distributions of 5-district genotypes of T. orientalis were observed for the examined farms. Our results indicate that at least 5 types of T. orientalis exist in Kumamoto and Okinawa prefectures of Japan and that genotype-specific PCR assays are highly applicable for the quarantine of transported cattle and for epidemiological surveys of bovine theileriosis in Japan.     

副结核分枝杆菌map0862基因的克隆及其在大肠杆菌中的表达

为探索MAP0862蛋白在分枝杆菌感染鉴别诊断中的作用,研究构建了MAP0862的原核表达map0862-pET32a(+)质粒,并对其进行了诱导表达。以副结核分枝杆菌P10基因组DNA为模板,经PCR方法扩增副结核分枝杆菌map0862基因片段,将其克隆到原核表达载体pET32a(+)中。将重组子转化到大肠杆菌BL21(DE3),在E.coli中诱导表达带组氨酸标签的map0862-pET32a(+)的融合蛋白。结果显示,经IPTG诱导表达出约55 kD的融合蛋白,用副结核阳性血清进行Western blot检测,证明MAP0862重组蛋白具有良好的免疫原性。     

The first survey of Theileria orientalis infection in Mongolian cattle

In the present study, we have surveyed the presence of a bovine Theileria protozoan, Theileria orientalis, in Mongolian cattle and engorging tick populations from selected provinces and districts in Mongolia. The percentages of infection in the cattle and ticks ranged from 8.8 to 66.6 and from 3.7 to 73.3, respectively, on a per district basis. The genetic diversity of T. orientalis isolates was also studied, based on the protozoan gene encoding a major piroplasm surface protein (MPSP). At least five genotypes (types 1, 3, 5, 7, and N-3) of T. orientalis were found to be circulating among the Mongolian cattle and tick populations. In particular, types 3 and N-3 were common in most of the districts examined, while a strong geographical relationship among the genotypes was not detected in the present study. This is the first epidemiological report describing the presence of T. orientalis infection in Mongolian cattle.     

ORF3 of duck circovirus: a novel protein with apoptotic activity

Duck circovirus (DuCV) is classified in the genus Circovirus of the Circoviridae family. Two major open reading frames (ORFs), encoding the replicase (ORF1/rep) and the capsid protein (ORF2/cap), have been recognized for DuCV. Sequence analysis show that another major conserved ORF (named ORF3) is located in the complementary strand of ORF1/rep of DuCV, and its function remains to be investigated. In this study, the ORF3 of DuCV was expressed in recombinant baculovirus-infected Sf9 cells. By IFA and Western blot analysis, the ORF3 protein was positive for the sera from ducks infected with DuCV. The percentages of apoptotic cells of the Sf9 cells infected with the recombinant baculovirus encoding ORF3 of DuCV were significantly higher than (P<0.05) that of the Sf9 cells infected with wild-type baculovirus at 24, 48 and 72 h postinfection. Based on our knowledge, we deduced that the ORF3 protein of DuCV might play an important role in viral pathogenesis via its apoptotic activity.     

牛分枝杆菌重组蛋白TB27.4的表达、纯化及活性鉴定

为探索TB27.4蛋白在牛结核病鉴别诊断中的作用,本试验以牛分枝杆菌Vallee Ⅲ株基因组DNA为模板,PCR扩增tb27.4全长基因片段,将其定向克隆到原核表达载体pET-32a(+)中,构建重组质粒pET-TB27.4,优化原核表达条件,并用AKTA Purifier对蛋白的纯化条件进行优化。SDS-PAGE结果显示重组蛋白为可溶性表达,且大小与理论值相符,用牛分枝杆菌阳性血清进行Western blotting检测有特异性条带,且可特异性地刺激牛分枝杆菌感染牛外周血淋巴细胞释放大量IFN-γ。结果表明,重组蛋白TB27.4具有良好的B细胞活性和T细胞刺激活性,为进一步研究其在牛结核病诊断中的作用奠定了基础。     

基因4型HEV上海株ORF_2编码蛋白表达及其抗原性分析

为了表达基因4型戊型肝炎病毒(HEV)上海株ORF2编码蛋白并分析其抗原性,采用套式RT-PCR方法扩增上海猪基因4型HEV代表株结构蛋白基因ORF2部分片段,构建含有该基因片段的pET30a(+)重组表达质粒,命名为pET-E;将重组表达质粒转化大肠杆菌BL21,经1mmol/L IPTG诱导得到表达,进行SDS-PAGE分析,然后用Western blot鉴定其抗原性,最后纯化蛋白。结果:SDS-PAGE证明该片段得到融合表达,分子量为33 ku。Western blot分析表明,重组蛋白可以与HEV阳性血清反应,表明该蛋白具有良好的抗原性;该融合蛋白含有组氨酸标签,可以用MagExtractor-His-tag-kit进行纯化。用纯化的重组蛋白作为包被抗原建立的间接ELISA检测89份猪血清样品,阳性率达80.4%,与已有的试剂盒检测结果相比,阳性符合率达95%。     

Molecular cloning, expression and first antigenic characterization of human astrovirus VP26 structural protein and a C-terminal deleted form

The open reading frame 2 (ORF2) of human astrovirus (HAstV) encodes the structural VP26 protein that seems to be the main antigenic viral protein. However, its functional role remains unclear. Bioinformatic predictions revealed that VP29 and VP26 proteins could be involved in virus-cell interaction. In this study, we describe for the first time the cloning and expression in Escherichia coli (E. coli) of a recombinant VP26 (rVP26) protein and a VP26 C-terminal truncated form (VP26 Delta C), followed by purification by NTA-Ni(2+) agarose affinity chromatography. Protein expression and purification were evaluated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (WB). Then, the purified proteins were evaluated for antigenic properties in enzyme linked immunosorbent assay (ELISA) using a polyclonal antibody (PAb) and a neutralizing monoclonal antibody (nMAb) named PL2, both of them directed to HAstV. The results presented herein indicate that the C-terminal end of the VP26 protein is essential to maintain the neutralizing epitope recognized by nMAb PL2 and that the N-terminus of VP26 protein may contain antigenic lineal-epitopes recognized by PAb. Thus, these recombinant proteins can be ideal tools for further antigenic, biochemical, structural and functional VP26 protein characterization, in order to evaluate its potential role in immunodiagnosis and vaccine studies.     

禽腺病毒血清4型Fiber2蛋白单克隆抗体制备及其抗原表位鉴定

旨在制备禽腺病毒血清4型（FAdV-4）纤突蛋白（Fiber2）的特异性单克隆抗体（MAb）,本研究将原核表达的可溶性重组蛋白NusA-Fiber2作为免疫原免疫BALB/c雌鼠,筛选获得3株能稳定分泌抗FAdV-4 Fiber2蛋白MAb的杂交瘤细胞株2G5、2G8、4C2,取细胞株2G5制备腹水并纯化,利用间接免疫荧光试验（immunofluorescence assay,IFA）和Western blot鉴定该单抗的特异性。用制备的单抗包被酶标板,通过一系列的优化,建立了FAdV-4 Fiber2双抗夹心ELISA检测方法。通过逐步截短分析鉴定出单克隆抗体识别的抗原表位区域。结果表明：成功获取3株单克隆细胞株2G5、2G8、4C2。MAb 2G5可与原核表达纯化的Fiber2蛋白及FAdV-4特异性反应。建立的双抗夹心ELISA检测方法具有很好的特异性、灵敏性和重复性。该MAb识别的表位序列是N端aa1—33。本研究成功制备了具有良好Western blot和IFA反应原性的单克隆抗体,为Fiber2蛋白功能研究和FAdV-4新型表位疫苗商品化研发奠定了基础。     

猪圆环病毒2型ORF3编码蛋白的体外表达

设计特异引物,以猪圆环病毒2型(PCV-2)杭州株HZ0201的基因组DNA为模板,PCR扩增出ORF3基因,构建了pGEX-4T-1-ORF3原核表达载体和pEGFP-C2-ORF3真核表达载体。ORF3基因全长315 bp,编码105个氨基酸。SDS-PAGE、Western blot分析及真核PK15细胞转染结果显示:ORF3蛋白在大肠杆菌中以包涵体形式存在,分子量大小约为37.7 ku;ORF3重组蛋白在真核PK15细胞的细胞核和细胞浆都有表达,尤其在细胞核中表达量较高,且对细胞有一定的毒性。     

山羊痘病毒糖蛋白基因ORF112的原核表达及多克隆抗体的制备

为探索山羊痘病毒(GTPV)糖蛋白基因ORF112在疫苗和诊断中的应用,应用PCR技术扩增GTPV弱毒疫苗株AV 41ORF112基因,将其克隆到pET-32a载体,转化感受态细胞BL21,经IPTG诱导后获得与预期大小相符的约37ku的融合蛋白,为可溶性和包涵体表达。应用镍离子亲和树脂对可溶性表达的目的蛋白进行纯化,然后用纯化的融合蛋白免疫Balb/c小鼠,制备多克隆抗体。免疫荧光试验表明该多克隆抗体可以与GTPV反应,为GTPV新型疫苗和诊断试剂的研究奠定了基础。     

Comparison of cellular schizont, soluble schizont and soluble piroplasm antigens in ELISA for detecting antibodies against Theileria annulata

The efficacy and suitability of cellular schizont, soluble schizont and soluble piroplasm antigens was compared for detecting antibodies against Theileria annulata. Fifty bovine sera of known identity were evaluated in ELISA using the above mentioned antigens. Antibody titres of 1:100 to 1:51,200 were detected while using soluble piroplasm and cellular schizont antigen in ELISA. The titres ranged between 1:100 to 1:25,600 with the soluble schizont antigen. Soluble piroplasm antigen exhibited the highest antibody titres followed by cellular schizont and soluble schizont antigens. Cellular schizont antigen proved to be better than soluble schizont antigen for detecting anti-schizontal antibodies. Antibody titres obtained by the three antigens exhibited a good linear correlation amongst each other. The study showed that soluble piroplasm and cellular schizont antigens can be used successfully for detecting antibodies against piroplasm and schizont stages of T. annulata, respectively in bovine sera.