猪圆环病毒1型ORF2基因的克隆与序列分析

【研究目的】提取沙门氏菌鞭毛蛋白并鉴定其抗原性;【方法】选用鸭沙门氏菌经半固体培养基诱导鞭毛,经肉汤培养后,用酸裂解、经差速离心、硫酸铵沉淀,分离出粗制鞭毛蛋白。将粗制的鞭毛蛋白用SDS-PAGE和磷钨酸负染投射电镜观察,并包板做ELISA检测其抗原性;【结果】SDS-PAGE显示提取的鞭毛蛋白相对分子质量约为58.0KD,且纯度较高,抗原性较好,每1000ml培养液可获得32.8mg鞭毛蛋白;【结论】酸裂解法可获得高质量的鞭毛蛋白,抗原性较好,为禽副伤寒沙门氏菌病诊断方法的建立奠定了基础。     

Pertussis toxin S1 mutant with reduced enzyme activity and a conserved protective epitope

Pertussis toxin (PTX) is a major virulence factor in whooping cough and can elicit protective antibodies. Amino acid residues 8 to 15 of PTX subunit S1 are important for the adenosine diphosphate-ribosyltransferase activity associated with the pathobiological effects of PTX. Furthermore, this region contains at least a portion of an epitope that elicits both toxin-neutralizing and protective antibody responses in mice. The gene encoding the S1 subunit was subjected to site-specific mutagenesis in this critical region. A mutant containing a single amino acid substitution (Arg9----Lys) had reduced enzymatic activity (approximately 0.02% of control) while retaining the protective epitope. This analog S1 molecule may provide the basis for a genetically detoxified PTX with potential for use as a component of an acellular vaccine against whooping cough.     

重组轮状病毒多抗原表位的克隆、表达及植物表达载体的构建

为了研制基于表位的轮状病毒亚单位疫苗,用PCR的方法得到RV(Rotavirus RV)VP4的抗原表位编码序列,并人工合成了RV VP7的3个抗原表位编码序列,通用辅助性T淋巴细胞表位(PADRE)氨基酸序列和破伤风毒素(Tetanus toxin,TT)上的一个T细胞激活位点[1,2],各位点间以非极性的甘氨酸和脯氨酸分隔以保持各表位的相对独立性,使用搭桥法PCR(Over lapextension PCR)、酶切、连接等基因工程的方法将各个表位片段串联拼接在一起,组合成一条轮状病毒(RV)多表位抗原基因,将其转入原核载体质粒pTHioHis B,在大肠杆菌DH5a克隆,并在BL21中表达,表达蛋白经SDS-PAGE分析,相对分子量与预期结果相符;重组蛋白经轮状病毒快速检测试剂盒检测有抗原性。将此多表位抗原基因插入高效植物表达载体pE1802,为研制重组轮状病毒抗原表位亚单位疫苗、转基因植物口服疫苗做了前期准备。     

O型口蹄疫病毒VP1 T细胞、B细胞表位双拷贝基因与大肠杆菌LTB基因融合表达产物的免疫应答

[目的]为预防口蹄疫与幼畜腹泻、生产安全高效的基因工程联合疫苗提供试验依据。[方法]通过基因工程技术,把O型口蹄疫病毒VP1双拷贝21-40(20aa)与141-160(20aa)表位肽基因——2020VP1与产肠毒素大肠杆菌LTB连接到表达载体上,在大肠杆菌中表达出具有抗O型口蹄疫病毒与产肠毒素大肠杆菌双重作用的融合蛋白。[结果]运用基因工程技术构建了融合表达载体r2020-B-2020。转化宿主菌BL21(DE3)RIL后的表达产物经SDS-PAGE分析,结果显示,重组融合蛋白的分子量约为41 kD,表达量较高。动物实验表明,融合蛋白能够诱发兔体产生较强的抗FMDV中和抗体,免疫豚鼠在低浓度FMDV刺激下产生特异性T淋巴细胞增殖反应,说明融合蛋白可以诱导机体产生有效的抗FMDV细胞及体液免疫反应。融合蛋白能够与霍乱毒素CTB抗体特异结合,免疫雌鼠能够抵抗一定强度的大肠杆菌毒株攻击。[结论]融合蛋白可以同时诱导机体产生较好的抗FMDV及ETEC免疫应答反应,具有开发成为FMDV与ETEC基因工程疫苗的应用价值。     

金黄色葡萄菌5型荚膜多糖偶联鞭毛蛋白疫苗对小鼠乳腺炎模型的保护作用

[目的]金黄色葡萄菌5型荚膜多糖(CP5)分别与鼠伤寒沙门氏菌鞭毛蛋白Flagellin、牛血清白蛋白(BSA)进行了偶联,并在小鼠模型上比较了这2种偶联疫苗所引起的体液免疫和粘膜免疫反应。[方法]以鼠伤寒沙门氏菌鞭毛蛋白为载体蛋白,与CP5进行化学偶联,并在小鼠乳腺部位免疫,然后通过检测小鼠乳汁中的抗体分析其对小鼠的免疫保护作用。[结果]乳腺局部免疫后flagellin-CP5组在乳腺部位引起了sIgA为代表的粘膜免疫反应和IgG为主的体液免疫反应。CP5和鞭毛蛋白的偶联物能够诱导更高的体液免疫以及粘膜免疫反应。[结论]鞭毛蛋白具有较强的免疫佐剂作用。     

Human melanoma proteoglycan: expression in hybrids controlled by intrinsic and extrinsic signals

Human malignant melanoma cells express specific chondroitin sulfate proteoglycans (mel-CSPG) on the surface, both in vivo and in vitro. Melanocytes in normal skin show no detectable mel-CSPG but can be induced to express the antigen when cultured in the presence of cholera toxin and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Most other cell types do not express mel-CSPG either in vivo or in vitro. A study was designed to examine regulatory signals controlling mel-CSPG expression. The gene encoding mel-CSPG was mapped to human chromosome 15, and this chromosome was introduced into rodent cells derived from distinct differentiation lineages. Three types of mel-CSPG--expressing hybrids were found: (i) hybrids derived from human melanomas; (ii) hybrids derived from human cells that do not express mel-CSPG; and (iii) hybrids derived from human cells expressing mel-CSPG that are antigen-negative but that are induced to express mel-CSPG when cultured on extracellular matrix instead of plastic surfaces. Thus, mel-CSPG expression can be controlled both through intrinsic signals, provided by the differentiation program of the rodent fusion partner, and through extrinsic signals, provided by specific cell-matrix interactions.     

Role of prostaglandins and cAMP in the secretory effects of cholera toxin

The role of adenosine 3',5'-monophosphate (cAMP) and prostaglandins in the pathogenesis of experimental cholera was evaluated. Fluid accumulated in the rabbit intestinal loop model after 16 hours of incubation with cholera toxin, prostaglandin E1, or prostaglandin E2, but not with membrane-permeable derivatives of cAMP or forskolin. Dibutyryl cAMP triggered a small, transient intestinal fluid accumulation response by 4.5 hours; however, the fluid was completely absorbed by 9 hours. After exposure of intestinal loops to cholera toxin, prostaglandin E was released into the intestinal lumen in a concentration-dependent manner independent of cAMP. Thus, not only cAMP, but also prostaglandins may regulate water and electrolyte secretion in cholera.     

Convergence of the secretory pathways for cholera toxin and the filamentous phage, CTXphi

Virulence of Vibrio cholerae depends on secretion of cholera toxin (CT), which is encoded within the genome of a filamentous phage, CTXphi. Release of CT is mediated by the extracellular protein secretion (eps) type II secretion system. Here, the outer membrane component of this system, EpsD, was shown to be required for secretion of the phage as well. Thus, EpsD plays a role both in pathogenicity and in horizontal transfer of a key virulence gene. Genomic analysis suggests that additional filamentous phages also exploit chromosome-encoded outer membrane channels.     

脑心肌炎增强型绿色荧光蛋白嵌合病毒的构建

携带增强型绿色荧光蛋白(Enhanced green fluorescent protein, EGFP)的脑心肌炎(Encephalomyocarditis virus, EMCV)嵌合病毒是研究该病毒体内外生物学特性的有力工具。因此,本研究基于前期构建的巨细胞病毒(Cytomegalovirus, CMV)感染性克隆,在EMCV基因组2A蛋白序列之后插入EGFP基因片段,并将重组质粒转染BHK-21细胞,获得携带EGFP的嵌合病毒。通过荧光定量PCR(real-time PCR)、间接免疫荧光(Indirect immunofluorescence assay, IFA)及半数组织细胞感染量测定(Median tissue culture infective dose, TCID₅₀)等方法对拯救病毒的基因组复制、蛋白表达及病毒粒子的感染性进行测定,结果证实嵌合病毒能够在BHK-21细胞上成功表达EGFP并产生完整的病毒粒子。然而,相较于野生型亲本拯救病毒,嵌合病毒在BHK-21细胞上的复制能力降低,并且在连续传代后逐渐失去绿色荧光信号,表明嵌合病毒中EGFP...     

转燕麦噬酸菌蛋白激发子基因flagellin的链霉工程菌株构建及鉴定

利迪链霉菌A01可以产生大量的抗生素——纳他霉素,其通过结合病原真菌细胞膜上的甾醇分子而起到抑制病原菌生长的作用.燕麦噬酸菌的鞭毛蛋白组分FLAGELLIN可以诱导植物抗性,激发活性氧及水杨酸防御反应途径.本研究将燕麦噬酸菌蛋白激发子基因flagellin克隆,接合转移入利迪链霉菌A01中,使工程菌增加诱导植物抗性的功能.PCR验证表明:成功获得了含flagellin基因的阳性工程菌株,证明通过链霉工程菌构建可实现抗生与诱抗的协同作用,预计会提高防治植物病害的效果.     

Pertussis toxin gene: nucleotide sequence and genetic organization

The current pertussis vaccines, although efficacious, in some instances produce undesirable side effects. Molecular engineering of pertussis toxin, the major protective antigen, could provide a safer, new generation of vaccines against whooping cough. As a first critical step in the development of such a vaccine, the complete nucleotide sequence of the pertussis toxin gene was determined and the amino acid sequences of the individual subunits were deduced. All five subunits are coded by closely linked cistrons. A promoter-like structure was found in the 5'-flanking region, suggesting that the toxin is expressed through a polycistronic messenger RNA. The order of the cistrons is S1, S2, S4, S5, and S3. All subunits contain signal peptides of variable length. The calculated molecular weights of the mature subunits are 26,024 for S1, 21,924 for S2, 21,873 for S3, 12,058 for S4, and 11,013 for S5. Subunits S2 and S3 share 70% amino acid homology and 75% nucleotide homology. Subunit S1 contains two regions of eight amino acids homologous to analogous regions in the A subunit of both cholera and Escherichia coli heat labile toxins.     

Reinitiation of growth in senescent mouse mammary epithelium in response to cholera toxin

Several lines of mouse mammary tissue that had been serially transplanted until mitotic senescence was reached were exposed in vivo to plastic implants that slowly released cholera toxin. Gland tissue surrounding the implants displayed new end buds, indicating reinitiation of growth and morphogenesis. The ability of cholera toxin, which elevates intracellular adenosine 3',5'-monophosphate, to temporarily reverse the senescent phenotype suggests that this mitotic dysfunction results not from generalized cellular deterioration but from specific changes in cell regulation.     

Divergence of quaternary structures among bacterial flagellar filaments

It has been widely assumed that the atomic structure of the flagellar filament from Salmonella typhimurium serves as a model for all bacterial flagellar filaments given the sequence conservation in the coiled-coil regions responsible for polymerization. On the basis of electron microscopic images, we show that the flagellar filaments from Campylobacter jejuni have seven protofilaments rather than the 11 in S. typhimurium. The vertebrate Toll-like receptor 5 (TLR5) recognizes a region of bacterial flagellin that is involved in subunit-subunit assembly in Salmonella and many other pathogenic bacteria, and this short region has diverged in Campylobacter and related bacteria, such as Helicobacter pylori, which are not recognized by TLR5. The driving force in the change of quaternary structure between Salmonella and Campylobacter may have been the evasion of TLR5.     

Three-dimensional structure of cholera toxin penetrating a lipid membrane

Two-dimensional crystals of cholera toxin bound to receptors in a lipid membrane give diffraction extending to 15 A resolution. Three-dimensional structure determination reveals a ring of five B subunits on the membrane surface, with one-third of the A subunit occupying the center of the ring. The remaining mass of the A subunit appears to penetrate the hydrophobic interior of the membrane. Cleavage of a disulfide bond in the A subunit, which activates the toxin, causes a major conformational change, with the A subunit mostly exiting from the B ring.     

Sequence of the alpha subunit of photoreceptor G protein: homologies between transducin, ras, and elongation factors

A bovine retinal complementary DNA clone encoding the alpha subunit of transducin (T alpha) was isolated with the use of synthetic oligodeoxynucleotides as probes, and the complete nucleotide sequence of the insert was determined. THe predicted protein sequence of 354 amino acids includes the known sequences of four tryptic peptides and sequences adjacent to the residues that undergo adenosine diphosphate ribosylation by cholera toxin and pertussis toxin. On the basis of homologies to other proteins, such as the elongation factors of protein synthesis and the ras oncogene proteins, regions are identified that are predicted to be acylated and involved in guanine nucleotide binding and hydrolysis. Amino acid sequence similarity between T alpha and ras is confined to these regions of the molecules.     

猪多杀性巴氏杆菌5:A型Ts-8株psl基因的克隆和序列分析

根据禽多杀性巴氏杆菌psl基因序列,设计了1对引物,从猪源多杀性巴氏杆菌5:A型Ts-8株中扩增出453bp的psl基因,将其克隆到pMD18-T载体后测序。序列分析表明,该基因与禽多杀性巴氏杆菌T16株的psl基因序列只相差2个碱基,同源性高达99%;与流感嗜血杆菌编码P6蛋白的基因序列同源性为83%。猪psl基因的推导氨基酸序列与禽psl基因、P6蛋白的氨基酸序列同源性分别为100%,87%。结果表明,多杀性巴氏杆菌psl基因在猪和禽的菌株中是高度保守的,而且与P6蛋白的氨基酸序列具有较高的同源性。     

检测鸡蛋中沙门氏菌的LAMP方法的建立及初步应用

根据GenBank上公布的沙门氏菌invA基因序列中的保守区域,设计一套环介导等温扩增(LAMP)引物,将其用于沙门氏菌的检测,结果成功地扩增出特异性的梯形条带。LAMP方法检测沙门氏菌纯培养的灵敏度为1.04×102 cfu.mL-1,对11株不同细菌进行LAMP检测,仅沙门氏菌获得阳性结果。应用该方法对扬州市场上销售的鸡蛋进行沙门氏菌检测,检测结果与传统培养方法相符合。因此,LAMP方法检测沙门氏菌具有灵敏度高,特异性强,耗时短,方法简便等特点,有望发展成为快速检测鸡蛋中沙门氏菌的有效手段。