3株猪源大肠埃希氏菌耐药基因的初步定位

本试验旨在对临床分离的猪源大肠埃希氏菌耐药基因进行初步定位。采用常规细菌分离培养、16S rRNA PCR扩增和序列测定方法从江西省3个规模化猪场送检的子宫脓液中分离鉴定病原菌,并通过质粒提取、转化大肠埃希氏菌DH5α感受态细胞及药敏试验对临床分离株的耐药基因进行初步定位。结果显示,分离鉴定到3株大肠埃希氏菌,其中JX-22分离株仅对氧氟沙星、大观霉素敏感,JX-26分离株仅对链霉素、氧氟沙星等4种药物敏感,JX-28分离株仅对氧氟沙星等3种药物敏感,均为多重耐药菌;3株大肠埃希氏菌均可纯化到分子质量大小不一的质粒。分离株、质粒转化菌及大肠埃希氏菌DH5α感受态细胞药敏试验对比结果显示,3株大肠埃希氏菌的耐链霉素、林可霉素、甲硝唑、氨苄西林、阿莫西林、大观霉素、丁胺卡那基因,JX-22和JX-26分离株的耐多西环素、氟苯尼考和复方新诺明基因,JX-22分离株的耐头孢曲松基因,JX-28分离株的耐头孢曲松、头孢噻肟、诺氟沙星基因均定位于细菌质粒上;JX-28分离株的耐多西环素、氟苯尼考和复方新诺明基因,JX-22分离株的耐诺氟沙星基因和JX-26分离株的耐头孢曲松、头孢噻肟基因均定位于其染色体上;3株分离株均无氧氟沙星耐药基因。本试验初步确定3株多重耐药猪源大肠埃希氏菌的大部分耐药基因定位于质粒上,为进一步研究猪源大肠埃希氏菌的耐药机理和有效控制措施奠定基础。     

鸡源大肠杆菌强毒株耐药基因的定位及耐药质粒消除

本试验对临床分离的多重耐药鸡源致病性大肠杆菌强毒株的耐药基因进行初步定位,为临床选择合适的治疗策略提供理论依据。从送检病死鸡的肝脏、心脏中分离鉴定致病菌,质粒提取试剂盒提取分离菌的耐药质粒,转化入基因工程菌JM109,通过质粒纯化、电泳和药敏试验对耐药基因进行了初步定位。并用艾叶水煮液对该菌株进行体外耐药质粒消除试验。结果分离鉴定到1株强毒力鸡源大肠杆菌,该菌呈多重耐药性,且仅对氟奇霉素和链霉素敏感;由质粒转化和药敏试验结果可初步将耐环丙沙星、青霉素、氧氟沙星、氟哌酸、林可霉素和复方新诺明的基因定位于耐药质粒上,并可随质粒的转移而使转化菌获得耐药性;用艾叶水煮液可使该菌的耐药质粒消除率达60%;质粒消除菌的药敏试验结果表明,消除耐药质粒的细菌恢复了对环丙沙星、青霉素、氧氟沙星、氟哌酸、林可霉素和复方新诺明的敏感性。本研究结果表明,分离菌的耐药基因分别位于质粒和染色体上,艾叶对耐药质粒有较强的消除作用,可作为临床治疗用药。     

大肠埃希氏菌-胸膜肺炎放线杆菌穿梭载体构建

合成胸膜肺炎放线杆菌质粒pKMA2425上长度为126bp及2214bp的2个DNA片段,克隆到大肠埃希氏菌质粒pGSI中,获得重组质粒pGSIA。PCR扩增pTKRED上的大观霉素抗性基因及线性化的pGSIA（126bpDNA片段-pGSI-2214bpDNA片段）,连接,电转化胸膜肺炎放线杆菌,在大观霉素的选择压力下筛选鉴定到连接正确的质粒,命名为pGSIAS。pGSIAS测序结果符合预期。在大观霉素的选择压力下,含有pGSIAS的胸膜肺炎放线杆菌血清1-10型菌株均生长良好;在氨苄青霉素的选择压力下,含有pGSIAS的大肠埃希氏菌生长良好。结果表明本研究构建的大肠埃希氏菌-胸膜肺炎放线杆菌穿梭载体pGSIAS序列正确,在胸膜肺炎放线杆菌及大肠埃希氏菌中均能复制并表达质粒上的相关基因。     

卵泡抑制素与GFP融合基因疫苗pEGISI的构建及表达

为构建卵泡抑制素与绿色荧光蛋白(GFP)融合基因疫苗pEGISI,将pcISI中的乙肝表面抗原(HBsAg)S基因及插入S基因中的抑制素(INH)基因片段酶切回收;PCR扩增pcISI中INH基因片段,调整阅读框,一起融合到pEGFP-N1中EGFP基因的5'端,构建ISI-EGFP融合表达质粒pEGISI。酶切和测序鉴定表明,重组质粒pEGISI构建成功。将pEGISI转染293T细胞,16h后检测到绿色荧光,48h检测到强烈荧光,说明融合基因在293T细胞获得高表达;ELISA检测证实,表达产物ISI-EGFP融合蛋白具有INH的抗原抗体反应原性。质粒pEGISI的成功构建及表达为抑制素基因免疫的机理及安全性研究奠定了基础。     

Plasmid-mediated florfenicol resistance in Mannheimia haemolytica isolated from cattle

The aim of this study was to analyse a florfenicol-resistant Mannheimia haemolytica isolated from a calf to determine the genetic basis of its florfenicol-resistance. The antimicrobial susceptibility and plasmid content of the isolate were determined. A florfenicol resistant plasmid carrying the floR gene was identified by PCR and transformed into Escherichia coli JM109 and HB101 strains. The plasmid was then mapped and sequenced completely. The isolate was resistant to chloramphenicol, florfenicol, oxytetracycline, kanamycin, dihydrostreptomycin, nalidixic acid, ampicillin, and amoxicillin; it carried a floR plasmid of 7.7kb, designated pMH1405. The mobilisation and replication genes of pMH1405 showed extensive similarity to the 5.1-kb pDN1 plasmid from Dichelobacter nodosus and the 10.8-kb pCCK381 plasmid from Pasteurella multocida. An adjacent 2.4-kb segment was highly homologous to the TnfloR region of the E. coli BN10660 plasmid. A plasmid-mediated floR gene was responsible for florfenicol resistance in the bovine respiratory tract pathogen M. haemolytica. The pMH1405 plasmid is the smallest floR-carrying plasmid reported to date. To the best of our knowledge, this is the first report of a florfenicol-resistant gene in M. haemolytica.     

Identification of canine transmissible venereal tumor cells using in situ polymerase chain reaction and the stable sequence of the long interspersed nuclear element.

Canine transmissible venereal tumor (CTVT) is a unique tumor that can be transplanted across the major histocompatibility complex (MHC) barrier by viable tumor cells. In dogs, CTVT grows progressively for a few months and then usually regresses spontaneously. A long interspersed nuclear element (LINE) insertion is found specifically and constantly in the 5' end of the CTVT cell c-myc gene, outside the first exon. The rearranged LINE-c-myc gene sequence has been used with polymerase chain reaction (PCR) to diagnose CTVT. However, in CTVT cells, the total length of the inserted LINE gene is not constant. In this experiment, variation in the inserted LINE gene was studied to determine which parts of the LINE sequence can be used as primers to identify CTVT cells with in situ PCR (IS PCR). The LINE gene was inserted between the TATA boxes in the promoter region of c-myc. In CTVT cells, deletions of different lengths are frequent in this gene. However, the 550-bp segment at the 5' end of the LINE-c-myc gene was stable. Thus, primers were designed to cover the stable 0.55-kb segment from the 5' end outside the first exon of the c-myc gene to the 5' end of LINE gene stable segment. With these primers and IS PCR, individual CTVT cells in formalin-fixed tissue sections and CTVT cultures were identified. Cells from other canine tumors were negative for this gene. In addition, the CTVT-specific, 0.55-kb segment was not found in any spindle-shaped cells from progressive or regressive phase CTVT. The IS PCR technique also did not detect any positive spindle-shaped cells in CTVT cell cultures. Thus, fibroblastic terminal differentiation is less likely to be a mechanism for spontaneous regression of CTVT cells.     

Correction of the Mutant SLA-1-632-TPK Gene from ToPigs Pig and Construction of SLA-1-TPK/pMD19-T Recombinant Plasmid

The swine leukocyte antigen (SLA) genes in pigs were the important immune gene group in antigen presentation, and studing on SLA could provide the references for the prevention of some infectious diseases. Earlier studies found that SLA-1-632-TPK gene in ToPigs pig had a deleted base in its coding sequence (a single base "C" was lost in 632 bp from the 5' end of the SLA-1-632-TPK gene), which lead to frameshift mutation. In order to correct the SLA-1-632-TPK gene, two pairs of gene-correction's primers were designed to correct the gene by the splicing overlap extension PCR (SOE-PCR) in template of recombinant plasmid of SLA-1-632-TPK/pMD18-T. Firstly,the 5'and 3'ends of SLA-1-632-TPK gene were amplified, respectively, then both of them were spliced and amplified to form an intact SLA-1-632-TPK gene. After detected by agarose electrophoresis, the interest of the product was further cloned into pMD19-T Simple vector. The positive clones were screened by colony PCR and then sequenced. The result showed that the 5'and 3' ends of the SLA-1-632-TPK gene were all amplified successfully by SOE-PCR, with the products of about 650 and 590 bp, which were consistent with the theoretical value of 648 and 585 bp, respectively. After spliced, the intact sequence of SLA-1-632-TPK gene was obtained with the product of about 1 200 bp, which was close to the theoretical value of 1 223 bp. The colony PCR result showed that the corrected gene was successfully inserted into pMD19-T Simple vector . After the sequence was analyzed by GENETYX version 9.0, it was shown that the nucleotide "C" in 632 bp numbered from the 5'end of the gene was added and the SLA-1-632-TPK gene was coded correctly. In this study, the SLA-1-632-TPK was corrected successfully, and the recombinant plasmid SLA-1-TPK/pMD19-T was constructed, which would lay a foundation to further study the protein expression and associated function of SLA-1-TPK.     

托佩克猪SLA-1-632-TPK基因的矫正及重组质粒SLA-1-TPK/pMD19-T的构建

猪白细胞抗原（swine leukocyte antigen,SLA）在猪免疫系统中起递呈抗原作用,对其展开研究可为猪相关传染病的预防提供依据。研究发现,托佩克猪SLA-1-632-TPK基因编码序列发生碱基缺失（距离SLA-1-632-TPK基因5'端632 bp处丢失1个碱基"C"）,导致移码突变。为矫正SLA-1-632-TPK基因,设计2对矫正引物,以原重组质粒SLA-1-632-TPK/pMD18-T为模板,利用剪切重叠延伸PCR（splicing overlap extention PCR,SOE-PCR）技术分别扩增SLA-1-632-TPK 5'和3'端,之后进行拼接,最后扩增全序列从而矫正目的基因,并进一步连接pMD19-T Simple载体,通过单菌落PCR筛选阳性克隆并测序,并通过GENETYX version 9.0软件对所测序列进行分析。结果显示,SOE-PCR成功扩增得到5'和3'端片段,大小约为650和590 bp,与理论设计值大小（648和585 bp）接近,经过拼接以后,得到全长约1 200 bp,与理论设计值1 223 bp接近。菌落PCR结果显示,矫正基因成功克隆入pMD19-T Simple载体。序列分析结果显示,托佩克猪SLA-1-632-TPK基因距离5'端632 bp处丢失的碱基"C"得到矫正并正确编码。本研究成功矫正了SLA-1-632-TPK基因,并构建其重组质粒SLA-1-TPK/pMD19-T,为下一步研究SLA-1-TPK蛋白表达和相关功能奠定基础。     

水貂沙门氏菌的分离鉴定及药物敏感性分析

为指导临床合理用药并为治疗患病水貂提供依据,本试验对山东省某水貂养殖场送检的7只疑似患有沙门氏菌病的病死水貂的肝脏和脾脏样品进行细菌分离鉴定及药物敏感性分析,通过分离纯化方法从样品中分离菌株,并采用革兰氏染色、生化鉴定和PCR方法对分离菌株进行鉴定。运用K-B药敏法检测菌株对临床常用药物的敏感性,并通过PCR方法检测菌株耐药基因及Ⅰ类整合子的携带情况。结果显示,本试验分离得到的7株菌均为革兰氏阴性、短小的杆菌;生化反应检测显示,分离菌株葡萄糖、麦芽糖、甘露醇、MR试验、枸橼酸盐、硫化氢试验均表现为阳性,初步鉴定分离菌株为沙门氏菌;PCR产物测序结果进一步表明7株分离菌均为沙门氏菌;药敏试验结果显示,7株沙门氏菌对头孢曲松、左氧氟沙星、氟苯尼考和多黏菌素较敏感,对氨基糖苷类药物、四环素和氨苄西林表现为耐药;耐药基因检测结果显示,7株沙门氏菌共检测出8种耐药基因blaTEM-1、blaCTX-M-1G、aadA1、aac(3′)-Ⅳ、aac(3′)-Ⅱc、aph(4′)-Ⅰa、aph(3′)-Ⅶ和oqxAB,以及基因盒为aadA1、arr-3-aacA4和blaPSE-1的Ⅰ类整合子。综上可知,本试验在送检的7只患病水貂的肝脏和脾脏样品中分离到7株沙门氏菌,分离菌株均为耐药菌株,且主要表现为多重耐药现象;耐药基因呈多样性,且存在位于质粒上的耐药基因扩大了耐药基因的传播范围,增加了细菌的耐药性,为临床用药治疗带来困难。     

Virulence of Streptococcus canis from canine streptococcal toxic shock syndrome and necrotizing fasciitis

The recent recognition of streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis (NF) in dogs caused by Streptococcus canis highlights our lack of knowledge regarding the mechanisms of virulence of this organism. Fifteen isolates of S. canis from cases of canine STSS and/or NF were examined for the presence of 10 Streptococcus pyogenes-associated virulence genes by Southern hybridizations using gene probes generated by PCR. The isolates lacked DNA with homology to eight of the 10 gene probes (speA, speB, speC, mf, ssa, scp, hasA, ska) under low stringency conditions. Thirteen and 15 of 15 isolates hybridized with streptolysin O and M protein gene probes, respectively. Twelve of 15 S. canis isolates were resistant to phagocytosis in canine blood. Electron microscopy revealed the presence of proteinaceous cell surface fibrillae. These results suggest that S. canis possesses M proteins and encodes streptolysin O, but lacks some of the other recognized virulence genes with significant homology to those in S. pyogenes.     

禽致病性大肠杆菌中四种抗氨基糖苷类药物耐药基因的分子流行病学调查

氨基糖苷类药物曾经是临床最为常用而有效的抗生素,长期应用与不合理使用使得该药物效果不尽理想。本研究参照GenBank中耐药基因相关序列,设计引物,结果从禽源大肠杆菌基因组中扩增出4种抗氨基糖苷类药物基因aadA1、strA、strB、aph(3′),经pGEX-T-easy和pET32a载体克隆和序列分析,证明扩增获得的基因序列与GenBank中参考序列同源性达到97%以上。对分离保存的216株禽致病性大肠杆菌进行氨基糖苷类药物耐药基因的分子流行病学检测,结果表明:aadA1阳性率高达49.1%,strA和strB的阳性率分别为56%和65.7%,aph(3′)的阳性率为16.2%;近三分之二的被检菌株携带有2种以上氨基糖苷类药物耐药基因。药敏试验结果显示所有被检菌株对链霉素耐药率为68.9%,而对阿米卡星的耐药率为38.9%。本研究结果说明禽类细菌的耐药性与相关耐药基因的检出率基本呈正相关,临床日益严重的耐药现象与耐药基因的普遍存在有着很大的关系,提示控制禽源耐药细菌对人类健康与卫生安全有重要意义。     

Characterization of antimicrobial resistance of Salmonella Newport isolated from animals, the environment, and animal food products in Canada

Multi-drug-resistant (MDR) Salmonella enterica serovar Newport strains are increasingly isolated from animals and food products of animal origin and have caused septicemic illness in animals and humans. The purpose of this study was to determine the occurrence and the epidemiologic, phenotypic, and genotypic characteristics of S. Newport of animal origin that may infect humans, either via the food chain or directly. During the 1993-2002 period, the Office International des Epizooties Reference Laboratory for Salmonellosis in Guelph, Ontario, received 36 841 Salmonella strains for serotyping that had been isolated from animals, environmental sources, and food of animal origin in Canada. Of these, 119 (0.3%) were S. Newport. Before 2000, none of 49 S. Newport strains was resistant to more than 3 antimicrobials. In contrast, between January 2000 and December 2002, 35 of 70 isolates, primarily of bovine origin, were resistant to at least 11 antimicrobials, including the extended-spectrum cephalosporins. The blaCMY-2', flo(st'), strA, strB, sulII, and tetA resistance genes were located on plasmids of 80 to 90 MDa that were self-transmissible in 25% of the strains. Conserved segments of the integron 1 gene were found on the large MDR-encoding plasmids in 3 of 35 strains additionally resistant to gentamicin and spectinomycin or to spectinomycin, sulfamethoxazole-trimethoprim, and trimethoprim. Resistance to kanamycin and neomycin was encoded by the aphA-1 gene, located on small plasmids (2.3 to 6 MDa). The increase in bovine-associated MDR S. Newport infections is cause for concern since it indicates an increased risk of human acquisition of the infection via the food chain.     

四川地区牦牛源肺炎克雷伯氏菌的分离鉴定及其耐药性分析

为了分析四川地区牦牛源肺炎克雷伯氏菌耐药性并确定其耐药基因的携带情况，本试验采集了四川地区不同养殖场患呼吸道疾病牦牛肺脏、咽拭子、鼻拭子共127份病料组织，分离得到了43株肺炎克雷伯氏菌，并采用微量肉汤稀释法结合PCR扩增法检测43株肺炎克雷伯氏菌的耐药性和耐药基因携带情况。结果表明：43株肺炎克雷伯氏菌对氨苄西林、阿莫西林、多西环素、磺胺间甲氧嘧啶4种药物耐药率较高，为65.12%~90.70%；对阿米卡星、氟苯尼考、多黏菌素B、大观霉素4种药物的耐药率在20.93%~48.84%之间；对头孢噻呋、恩诺沙星、环丙沙星3药物的耐药率在18.60%~25.58%之间；多数筛选分离得到的菌株均呈现出多重耐药性，耐9、10种药物的菌株最多，分别占分离菌株的23.3%和20.9%。通过使用PCR扩增法检测分离菌携带的耐药基因情况，结果表明：bla_TEM、bla_SHV、sul2、sul3、floR 5种耐药基因检出率较高，检出率在62.8%~69.8%之间；ant （3″）-Ⅰa、aph （3'）-Ⅱa、aac （6'）-Ⅰb、aacC2 4种耐药基因检出率较低，检出率在7.0%~20.9%；bla_CTX-M、Mcr-1、qnrA、qnrS、rmtB 5种耐药基因未检出。综上所述，本试验中分离得到肺炎克雷伯氏菌具有较强的耐药性并携带有较多的耐药基因，为了更好的促进四川省牦牛养殖产业的发展，应注意避免滥用抗生素并通过药敏试验对症下药。     

Antimicrobial susceptibility of Enterococcus strains isolated from poultry faeces

We have investigated the resistance of Enterococcus isolated from poultry faeces to antibiotics commonly used as therapy of enterococcal infections. Identification was made by the method of Facklam and Collins. Minimal inhibitory concentrations of penicillin, ampicillin, vancomycin and teicoplanin were determined and high level aminoglycoside resistance was investigated. Genes codifying high level aminoglycoside resistance (HLAR) were determined by PCR. Fifty five Enterococcus strains were isolated (63.6% E. faecalis, 12.7% E. mundtii, 9.1% E. faecium, 7.3% E. casseliflavus, 3.7% E. durans and 3.6% E. hirae). None of the strains were resistant to VAN, TEC, P or AM. HLAR was found in 34.5% of strains for SM, 27.3% for KM and 7.3% for GM. The gene for the bifunctional enzyme was found only in one strain, that showed HLAR to GM and KM. Fourteen strains harboured the gene aph(3')-III, being 11 resistant to KM and STR, and three resistant to GM, KM and STR. The remaining six strains showed HLAR to STR, but were negative for the three genes tested by PCR. The gene ant(4'4") was not detected in any of the strains. No unexpected vancomycin resistance was detected. The resistance rates among poultry strains were lower than those found among human strains isolated from hospital patients in recent Canary studies.     

Characterization of a plasmid-encoded adhesin of an avian pathogenic Escherichia coli (APEC) strain isolated from a case of swollen head syndrome (SHS)

An avian pathogenic Escherichia coli (APEC) strain designated SHS4, isolated from a chicken with clinical signs of swollen head syndrome (SHS), adhered to but did not invade Hep-2 and tracheal epithelial cells. The PCR amplified fimA, csgA and tsh gene sequences. It produced Ia, Ib, E1, E3, K, and B colicins, but not colicin V and aerobactin. It harboured two plasmids of 60 and 98MDa and was resistant to streptomycin and tetracycline. Conjugation with a nalidixic acid (Na) resistant K-12 recipient strain (MS101) showed that the 98MDa plasmid did not transfer, whereas transfer of the 60MDa plasmid resulted in concomitant transfer of adhesion to Hep-2 and tracheal epithelial cells, production of the colicins Ia, E1, E3, and K, and the tsh-related DNA sequence. Transposon (TnphoA) mutagenesis of strain TR4 gave rise to strain Mut23, which lost its adhesive capacities, but was still able to express the same colicins as did strain TR4. PCR was able to amplify the tsh-related DNA sequence in this strain and a molecular probe based on transposon TnphoA indicated that the transposon was inserted in the 60MDa plasmid. Based on these results, we suggest that the 60MDa plasmid have adhesion genes, which may be responsible for the initial colonization of the upper respiratory tract of chickens.     

紫花苜蓿MsZIP基因超表达载体的构建及烟草转化

根据已经克隆得到的MsZIP基因(GenBank序列号:HQ911778),合成编码区cDNA,构建植物超表达载体PBI-MsZIP。将MsZIP基因插入到含有启动子35S和终止子nos的载体PBI121中, 酶切鉴定表明,目的基因已经正确的插入到载体中,超表达载体构建成功。采用CaCl₂冻融法将其转入农杆菌中,然后采用农杆菌介导的方法转化烟草(Nicotiana tobacum),共得到12株抗性烟草苗,选取其中长势良好的3株进行分析。结果表明:转基因烟草的PCR, RT-PCR和Southern-blot检测均得到了与目的条带大小一致的片段,说明已经成功获得了能够表达MsZIP基因的转基因烟草;进一步对转基因烟草进行组织化学染色分析,该基因在根、茎、叶中都可以表达。本试验为MsZIP基因功能的研究提供了理论依据和试验材料。     

鸡柔嫩艾美耳球虫不同抗药性虫株的种内多态性研究

应用RAPD技术进行柔嫩艾美耳球虫4个单一抗药性虫株与1个敏感株的基因组DNA多态性分析，发现4个抗药性虫株及敏感株之间的相似值均大于99%；OPAO3引物对抗盐霉素株扩增出一条约500bp的特异条带，OPH02引物对抗克球粉株和抗盐霉素株均扩增出了约1kb的第三条主带，这些特异条带的出现很可能与球虫抗药性基因有关，有可能用于抗药性虫株的诊断与鉴定。     

广东水禽源大肠埃希菌耐药性及氨基糖苷类耐药基因研究

为了调查广东地区水禽源大肠埃希菌对氨基糖苷类药物耐药现状和耐药基因的流行情况,探索大肠埃希菌的氨基糖苷类耐药基因型与耐药表型之间的关系,本研究采用琼脂梯度稀释法测定251株广东地区水禽源大肠埃希菌对氨基糖苷类药物的耐药性和采用PCR方法检测耐药基因。药敏试验结果显示,大肠埃希菌对链霉素耐药较严重,鸭源和鹅源的耐药率分别为81.58%和75.43%,对庆大霉素和卡那霉素较敏感,耐药率分别为25%和54.86%,对阿米卡星最为敏感,其中鸭源的菌株耐药率仅为1.32%。PCR方法检测显示,aadA1和aph(3′)-1检出率较高,为84.6%和91.9%,表明携带aadA1和aph(3′)-1耐药基因的水禽源大肠埃希菌在广东地区呈流行趋势。耐药基因型与耐药表型相关性分析结果显示,耐药基因rmtB的携带与4种药物(庆大霉素、阿米卡星、卡那霉素和壮观霉素)耐药株的产生具有显著相关性,表明耐药基因rmtB对大肠埃希菌耐药株的产生起重要的作用。本试验结果可为指导广东地区水禽大肠埃希菌病的临床用药提供理论基础和研究大肠埃希菌的耐药基因提供相关的数据。     

猪附红细胞体ENO基因重组腺病毒穿梭质粒的构建及真核表达

为构建猪附红细胞体ENO基因重组腺病毒穿梭质粒,试验根据GenBank中登录的猪附红细胞体ENO基因序列（登录号：CP002525.1）设计特异性引物,对ENO基因进行PCR扩增,并将纯化后的PCR产物克隆到pMD19-T载体中。用Kpn Ⅰ和Xho Ⅰ对pMD19T-ENO进行双酶切后,将其亚克隆至腺病毒穿梭载体PCR259中,构建PCR259-ENO重组质粒,提取重组质粒进行鉴定。应用脂质体介导转染法将鉴定正确的PCR259-ENO重组穿梭质粒转染293细胞,应用间接免疫荧光法（IFTA）检测ENO基因在293细胞中的表达。结果显示,试验克隆的ENO基因长为1 182 bp,编码393个氨基酸,与GenBank中ENO基因序列（登录号：CP002525.1）同源性为99%。构建的重组腺病毒穿梭质粒PCR259-ENO经PCR和酶切鉴定正确,并且能在293细胞中表达,表明ENO基因成功插入腺病毒穿梭质粒PCR259中,重组腺病毒穿梭质粒PCR259-ENO构建成功。     

PCV2衣壳蛋白羧基端基因在T4噬菌体表面的展示及ELISA方法的建立

用PCR扩增猪圆环病毒Ⅱ型广东分离株的衣壳蛋白羧基端基因,将PCR产物连接到pR质粒,转化DH5α细胞,筛选阳性克隆进行PCR鉴定并测序后,转化E2菌,将重组E2菌与缺陷型噬菌体T4-Z1同源重组后,得到重组噬菌体,经SDS-PAGE和Western blotting分析,表明衣壳蛋白片段在噬菌体表面正确展示,表达的融...