新疆出血热病毒S基因在真核系统的克隆和表达

目的　在真核系统表达XHFVS基因片段 ,制备安全、特异、便于操作的S蛋白。方法设计引物 ,扩增得到S基因编码片段 ,用杆状病毒表达载体pFastBacHTb克隆S基因 ,并在昆虫细胞中表达S基因。结果　地高辛探针杂交和PCR扩增结果表明成功地构建了含XHFVS基因片段的pBac Sxhf质粒。SDS PAGE分析表达产物在相对分子质量 (Mr)为 6 6× 10 3 下方出现 1条明显的蛋白条带 ,Westernblot发现该蛋白带可以与抗XHF病毒核蛋白单克隆抗体发生反应。结论　构建的含XHFVS基因的重组杆状病毒可以在昆虫细胞表达S蛋白 ,该蛋白可与特异性抗体相结合     

幽门螺杆菌oipA基因片段的克隆表达及抗原性分析

目的:分片段克隆表达幽门螺杆菌oipA基因,确定抗原性最强的重组蛋白肽段.方法:从Hp国际标准株NCTC11637中获取oipA全长基因,克隆至pGEM-T载体并鉴定正确,以重组pGEM-T/oipA为模板PCR扩增6个不同大小的oipA基因片段,与表达载体pET-42a连接,转化大肠杆菌BL-21,重组子经PCR、酶切、测序鉴定.IPTG诱导重组蛋白肽段的表达,经Western Breeze 化学发光法鉴定融合蛋白;优化诱导时间和IPTG浓度,诱导重组蛋白大量表达;Ni-NTA His*Bind树脂亲和纯化表达产物;羊抗Hp多克隆抗体,Western blot法检测纯化蛋白的抗原性,间接ELISA法筛选抗原性最强的重组肽段.结果:6条oipA基因片段在大肠杆菌中都得到了表达,表达的蛋白肽段均可被抗Hp多克隆抗体识别,具有抗原性,其中反应性最强的是最小的蛋白肽段.结论:oipA基因片段可在原核系统中表达,所获取的最小的肽段抗原性最强,可望制备检测血清相应抗体的试剂盒.     

Cloning and expression of the Pseudomonas aeruginosa exoenzyme S toxin gene

The gene for exoenzyme S, an ADP-ribosyl transferase, was cloned from Pseudomonas aeruginosa strain DG1 using an oligonucleotide probe based on the partial N-terminal amino acid sequence to screen a library of DG1 SstI fragments inserted into pKT230 in Escherichia coli DH1. A positive clone, designated pPD3, hybridized with the oligonucleotide probe and contained a 15 kb SstI insert. In E. coli minicells pPD3 expressed a single protein of Mr 68,000. This protein was localized primarily in the periplasm in E. coli. A 3.6 kb HindIII-BamHI fragment was subcloned into the vector pT7-4 which contains the promoter from bacteriophage T7 to construct pT7-4HB. In E. coli strains expressing the T7 RNA polymerase on a second plasmid, the Mr 68,000 protein was expressed and shown to react with antibodies to exoenzyme S. No enzymatic activity was detected in cell sonicates or culture supernatants of E. coli (pPD3). Cell sonicates of E. coli (pT7-4HB) however were cytotoxic to HeLa cells and this cytotoxicity was neutralizable with anti-exoenzyme S antiserm. Thus, exoenzyme S expressed in E. coli is toxic but not enzymatically active. When plasmids carrying the exoenzyme S gene were introduced into P. aeruginosa, there was a significant increase in ADP-ribosyl transferase activity, indicating that the plasmid encoded protein is enzymatically active in P. aeruginosa.     

SARS冠状病毒N蛋白的克隆与表达

目的　克隆和表达SARS冠状病毒N蛋白 ,并分析其免疫学活性。方法　采用RT PCR从SARS冠状病毒RNA中扩增出编码N蛋白的基因 ;经克隆和测序分析后 ,亚克隆至表达载体pGEX 4T 2 ,转化大肠杆菌JM10 9,PCR和双酶切鉴定 ;阳性菌株经IPTG诱导 ,SDS PAGE分析 ;大量诱导表达N蛋白 ,亲和层析予以纯化 ;免疫印迹分析纯化蛋白对SARS的诊断效果 ;用纯化的融合蛋白免疫小鼠观察其诱导的抗体应答。结果　RT PCR扩增出N蛋白基因的特异片段 ,获得的阳性克隆序列与Gen Bank中登录的SARS冠状病毒的N蛋白基因序列同源性为 99.8% ;N蛋白基因被亚克隆到表达载体pGEX 4T 2 ,在JM10 9中表达了N蛋白 ,表达的蛋白经亲和层析获得纯化 ;纯化蛋白能被SARS病人血清识别 ;免疫小鼠诱导产生了高滴度的抗体。结论　成功构建了SARS冠状病毒N蛋白的重组表达质粒 ,在大肠杆菌中表达的N蛋白融合蛋白具有良好的免疫学活性。     

Cloning the ends of size selected Sfi I fragments

As an initial step in the physical mapping of the fragile X region a library of Sfi I ends was constructed from the size class of human Sfi I DNA fragments, which includes the fragment with the locus DXS105. Since Sfi I recognizes the sequence GGCCNNNNNGGCC and leaves a 3 base indeterminate "sticky" end, we used a mixture of 64 synthetic deoxynucleotide oligomers to modify these ends for cloning. The oligomers were of the general form AATTNNN. Ligation of these heptamers to the indeterminate Sfi I ends converted them to the EcoR I sticky end. A suppressor tRNA gene was ligated onto this end as a selectable marker and the DNA was cloned in the lambda phage vector Charon 21A. Analysis showed that clones selected for the presence of the tRNA gene contained Sfi I ends. Because this library was constructed from a specific size class of fragments, it was very reduced in complexity. This will simplify the process of identifying the clone which contains the end of the DXS105 fragment. The use of this strategy for chromosome "jumping" is discussed.     

SARS-CoV受体ACE-2基因的克隆及在真核细胞中的表达

目的克隆、真核表达严重急性呼吸综合征冠状病毒(SARS-CoV)受体ACE2基因,建立安全、可靠的SARS-CoV保护性体液免疫评价体系。方法采用Trizol一步法提取人右心衰心房组织总RNA,逆转录巢式聚合酶链反应(RT-nested-PCR)扩增ACE2全长基因,克隆人pcDNA4/HisMax-TOPO表达载体,重组质粒转染293T细胞进行瞬时表达,Western Blot检测其真核表达;建立细胞融合抑制实验以检测SARS-CoV中和抗体,并与SARS-CoV假病毒中和试验进行平行比较。结果重组质粒可在真核细胞中表达ACE2蛋白,其蛋白表达细胞可与S蛋白表达细胞产生细胞融合现象,并可用于中和抗体的检测;其结果与SARS-CoV假病毒中和试验较为一致。结论克隆和真核表达了SARS-CoV受体ACE-2基因;基于其蛋白真核表达的细胞融合抑制实验可用于SARS-CoV中和抗体的检测。     

Cloning and expression of two human recombinant monoclonal Fab fragments specific for EBV viral capsid antigen

Serum titers of antibody to Epstein-Barr virus (EBV) viral capsid antigen (VCA) have been positively correlated with malignancies of lymphoid proliferation, such as Burkitt's lymphoma and Hodgkin's lymphoma. We have constructed a phage display combinatorial antibody Fab library from a patient with marginal zone B cell lymphoma associated with Sj?gren's syndrome and carrying high serum anti-EBV-VCA IgG titer. Fab fragments were selected by panning against EBV-VCA protein coated onto ELISA plates, and selected Fab clones were characterized by ELISA, western blotting (WB), indirect immunofluorescence assay and immunohistochemistry. We have established two Fab clones, Fab-aVCA1 and Fab-aVCA21, which specifically recognize EBV-VCA by ELISA and WB. Inhibition ELISA competition showed that both clones could significantly reduce the binding of specific anti-EBV-VCA mAb to its relevant proteins. Furthermore, these two Fab clones could localize VCA protein in the EBV-positive P3HR1 and Daudi cell lines, as well as in tissue samples from patients with EBV-infected lymphoid malignancies. These results indicate that our two Fab clones are novel human mAbs specific for EBV-VCA protein and may have potential benefits for development of novel diagnostic and therapeutic approaches in EBV-related lymphoid malignancies.     

Regulation of type I collagen gene expression in bone

The regulation of COL1A1 gene expression in bone was studied by measuring the activity of type I collagen promoter fusion genes (ColCAT) in permanently transfected osteoblastic cells and calvariae from transgenic animals. The basal activity of ColCAT fusion genes in transfected cells is mediated by DNA sequences between -3.5 to -2.3 kb while expression in vivo requires sequences between -2.3 and -1.7 kb. Parathyroid hormone, 1,25-dihydroxyvitamin D3 and interleukin-1 decrease the activity of ColCAT fusion genes in osteoblastic cells and transgenic calvariae. Because there may be differences between the expression of ColCAT fusion genes in cultured cells and intact bone, it will be important to compare data obtained from transfected cells with an in vivo model such as calvariae from transgenic mice.     

HBV Pre S2单链抗体基因在大肠杆菌中的克隆及初步表达

利用基因重组技术，在成功构建ＨＢＶ Ｐｒｅ Ｓ２单链抗体基因基础上，引人ＥｃｏＲ Ｉ和Ｓａｌ Ｉ酶切位点，克隆到原核高效表达质粒ｐＢＶ２２０中，筛选到２个重组克隆，经转染大肠杆菌ＤＨ５α，４２℃热敏诱导５～６小时后，超声裂解细菌产物在包被有ｐｒｅ Ｓ２抗原的阻断ＥＬＩＳＡ中，有明显的阻断亲本３Ｂ９单抗对ｐｒｅ Ｓ２抗原的亲和活性，在ＳＤＳ－ＰＡＧＥ中，分子量２．８×１０~４处显示一条表达产物带，表达产物占菌体总蛋白的４％左右。初步表明单链抗Ｐｒｅ Ｓ２基因抗体在大肠杆菌中表达，且有较好的Ｐｒｅ Ｓ２抗原结合活性。     

Ⅴ型腺病毒纤毛基因的克隆及其在大肠杆菌中的表达

目的克隆腺病毒纤毛基因,为构建腺病毒靶向性载体创造条件.方法应用限制性内切酶酶切技术,酶切腺病毒骨架质粒pAdEasy-1,经多次亚克隆最后完成克隆纤毛基因,并构建纤毛基因原核表达载体.用IPTG诱导纤毛蛋白的表达,再用SDS-PAGE和Western blot对其进行分析.结果成功地克隆纤毛基因.质粒pBS/Fiber经限制性内切酶酶切鉴定及测序证实了其正确性.经质粒pQE/Fiber转化的细菌,在IPTG诱导下可表达一种新的蛋白,并于5h表达量最高.经Western blot证实,在变性条件下表达蛋白的Mr为62 000,在非变性条件下为186 000.结论已克隆的纤毛基因能在大肠杆菌中表达,并具有三聚体结构,可用于腺病毒载体靶向性构建.     

Identification of canine coronavirus strains from feces by S gene nested PCR and molecular characterization of a new Australian isolate

A nested PCR (nPCR) assay for the detection of canine coronavirus (CCV) in fecal samples is described. The target sequence for the assay was a 514-bp fragment within the spike (S) glycoprotein gene. The sensitivity of the assay is extremely high, detecting as little as 25 50% tissue culture infective doses per g of unprocessed feces. A clinical trial using dogs challenged orally with CCV SA4 and CCV NVSL was used to compare viral isolation and the nPCR assay as detection techniques over a 2-week period of infection. Virus isolation detected CCV shedding from day 4 to 9 postchallenge, while the nPCR assay detected CCV shedding from day 4 to 13 postchallenge. Cloning and sequencing of the nPCR assay product enabled investigation of the evolutionary relationships between strains within the S gene. The simple and rapid procedure described here makes this assay an ideal alternative technique to electron microscopy and viral isolation in cell culture for detection of CCV shedding in feces. The described assay also provides a method of identifying new strains of CCV without the complicated and time-consuming practice of raising antibodies to individual strains. This is illustrated by the identification, for the first time, of an Australian isolate of CCV (UWSMN-1).     

Investigation into the causes of canine infectious respiratory disease: antibody responses to canine respiratory coronavirus and canine herpesvirus in two kennelled dog populations

Summary. Two training centres for working dogs were monitored for one year to determine the presence of viruses and viral antibodies and their association with canine infectious respiratory disease (CIRD). Tonsillar swabs and serum were obtained from dogs on entry into the kennels and in regular intervals thereafter. Additional samples were collected during outbreaks of CIRD. The swabs were examined by virus culture and PCR for canine parainfluenza virus, canine adenovirus, canine herpesvirus (CHV) and canine respiratory coronavirus (CRCoV). Furthermore the prevalence of antibodies to CHV and CRCoV was determined. During this study CIRD was reported mainly in one of the two kennels investigated. In that kennel antibody responses to CRCoV indicated a seasonal occurrence of the virus, which coincided with two outbreaks of respiratory disease. CHV antibody responses were detected throughout the year. In the other kennel, which reported few cases of CIRD a high prevalence of antibodies to CRCoV was detected on entry but only sporadic seroconversions to CRCoV or CHV. By PCR three dogs were found positive for CRCoV in one kennel whereas all PCR tests for other viruses were negative for both kennels. Virus culture failed to detect any viruses in either kennel.     

TEM-1型β-内酰胺酶编码基因的克隆表达

目的：构建TEM-1型β内酰胺酶基因的表达载体。方法：通过PCR扩增TEM—1全编码基因,将其连接入pBK—CMV载体中,并在大肠杆菌JM109中表达。采用琼脂二倍稀释法对TEM-1克隆菌株进行MIC检测,双纸片法筛选及确证实验检测其袁型,等电聚焦（IEF）电泳测定其等电点（pls）。结果：经酶切测序鉴定TEM—1基因的表达载体构建正确。重组菌产pl为5．4的TEM-1广谱酶,仅对青霉素类耐药。结论：TEM—1基因原核表达载体正确构建,为进一步研究打下了基础。     

HSP70基因的克隆与表达

目的 构建HSP70真核表达质粒以用于肾脏缺血预适应中HSP70作用及机制研究。方法用肝癌细胞系HepG2,用RT-PCR方法扩增HSP70cDNA,并利用T载体作为过渡,将HSP70基因克隆到真核表达载体pAAV-MCS中。重组质粒转染NIH3T3细胞,Western-blot法鉴定重组质粒HSP70／pAAV-MCS能在哺乳动物细胞中正确表达。结果RT-PCR方法正确地扩增出了全长HSP70基因。限制性内切酶酶切和测序结果证实HSP70基因克隆完全正确。重组质粒转染哺乳动物细胞系NIH3T3后,ECL-Western-blottimg法证实了目的基因能在其中正确表达。结论成功地克隆真核表达重组质粒HSP70／pAAV-MCS,为下一步研究HSP70在肾脏缺血预适应的作用及其机制打下基础。     

Aberrant type I and type III collagen gene expression in human breast cancer in vivo

Increased synthesis and degradation of extracellular matrix components are associated with breast cancer development. This study evaluated type I and type III procollagen mRNA expression and the corresponding protein synthesis and maturation, as well as the tissue distribution of these collagens, in benign breast lesions, infiltrating ductal carcinomas, and their metastases by in situ hybridization and immunohistochemistry. In the benign lesions, the type I and type III collagen bundles were regularly organized and the expression of the corresponding mRNA was weak, indicating a relatively slow collagen turnover. In the malignant tumours, increased expression of type I and type III procollagen mRNAs was observed in the fibroblastic cells of the stroma; the malignant epithelial cells did not participate. The staining of corresponding newly-synthesized pN-collagens showed aberrant bundles in the invasive front of the malignant tumours. Newly-synthesized type I and type III procollagens were occasionally observed in fibroblastic cells, particularly in grade 2 and grade 3 tumours. Metastases of breast carcinoma resembled poorly differentiated primary tumours with respect to their collagen synthesis and deposition. The increased synthesis of fibrillar type I and type III procollagens may serve as a pathway for tumour invasion. The enhanced synthesis is associated with the formation of aberrant collagen bundles, which may be more readily degradable and may thus facilitate breast tumour invasion.     

猪链球菌2型人源分离株截短的溶菌酶释放蛋白基因的克隆及原核表达

目的:克隆及表达猪链球菌2型(S.suis2)人源分离株Habb截短溶菌酶释放蛋白(MRP)基因。方法:根据S.suis2mrp基因的序列设计引物,克隆和分析江苏海安患者分离株Habb截短mrp基因(tmrp),构建原核表达质粒pGEX4T-2-mrp。在大肠杆菌中诱导带有谷胱甘肽转移酶(GST)标签的融合蛋白tMRP-GST表达;经亲和层析法纯化后,用凝血酶酶切去除重组蛋白中的GST,制备纯化的截短MRP(tMRP)抗原。用Westernblot检测重组抗原的活性。结果:序列分析表明,获得的tmrp基因长957bp;原核表达的融合蛋白tMRP-GST的相对分子质量(Mr)约61000;凝血酶处理的tMRP抗原的Mr约为35000。Westernblot分析显示,MRP-GST、tMRP蛋白均可与MRP多克隆抗血清发生特异性反应。结论:成功地克隆人源分离株Habb截短的mrp基因,并在原核系统实现高效表达,制备的纯化抗原tMRP具有良好的抗原性,为开展相关免疫学研究奠定了基础。