犬新孢子虫巨噬细胞转移抑制因子的原核表达

为筛选犬新孢子虫保护性抗原,克隆表达犬新孢子虫巨噬细胞转移抑制因子(MIF)并对其免疫原性进行分析。根据GenBank公布的犬新孢子虫MIF(NcMIF)序列,利用分子生物学软件设计2套特异性引物,以NcMIF mRNA反转录产物为模版,采用PCR扩增出NcMIF基因和2种突变基因NcMIFm和NcMIFhis,选用原核表达载体pET26b,分别构建重组质粒pET26b-NcMIF和pET26b-NcMIFm。重组质粒分别转入E.coli BL21(DE3)中进行表达,纯化后的蛋白利用免疫印迹法进行鉴定。结果显示,rNcMIF和rNcMIFm这2种重组蛋白在BL21(DE3)表达菌株中均为可溶性表达,诱导表达最佳条件为30℃诱导3h IPTG诱导浓度为1mmol/L;Western blot分析结果表明,该表达产物能与抗rNcMIF绵羊血清发生反应,说明获得的蛋白具有较好的抗原性和特异性。     

犬新孢子虫巨噬细胞转移抑制因子生物学特性鉴定

目的 通过原核系统表达犬新孢子虫巨噬细胞转移抑制因子(NcMIF),并对该蛋白的免疫调节作用进行分析。方法 通过GenBank发表的序列,利用分子生物学软件设计了一对特异性引物,通过RT-PCR方法扩增出NcMIF全基因,经测序分析后,将NcMIF亚克隆到原核表达载体pET28a(+),然后将鉴定为阳性的重组质粒转化到E. coli BL21(DE3)中用IPTG进行诱导表达。利用HPLC对可溶性表达的重组NcMIF蛋白进行纯化,去除内毒素,最后对NcMIF的免疫调节作用进行鉴定。结果 NcMIF没有明显的抗糖皮质激素的免疫抑制作用,也没有上调巨噬细胞TNF-α和NO表达量的作用。结论 实现了NcMIF在大肠杆菌系统中的可溶性表达并对其功能进行了鉴定,为进一步探究NcMIF生物学功能及其MIF在宿主免疫调节中的作用奠定基础。     

奥氏奥斯特他线虫巨噬细胞转移抑制因子克隆表达与酶功能鉴定

通过原核表达系统表达奥氏奥斯特他线虫（Ostertagiaostertagi）巨噬细胞转移抑制因子（OoMIF）,并对该蛋白的酶功能进行鉴定。通过GenBank和NematodeV3．0数据库发表的序列,利用分子生物学软件设计了1对特异的引物,通过RT—PCR扩增OoMIF全基因,经测序分析后,将OoMIF亚克隆到pET28a（＋）,然后将鉴定为阳性的重组质粒转化到BL21中用IPTG进行诱导表达。利用HPLC对可溶性表达的重组OoMIF蛋白进行纯化,同时通过免疫印迹对线虫自身的MIF及纯化后的OoMIF进行鉴定,最后对OoMIF的互变异构酶和氧化还原酶活性进行鉴定。结果表明OoMIF具有互变异构酶活性,但没有氧化还原酶活性。为进一步探究OoMIF生物学功能及其MIF在宿主免疫调节中的作用提供依据。     

柞蚕微孢子虫单克隆抗体的研制及诊断

研究报告了柞蚕微孢子虫Nosemaantheraeae(简称N .a)单克隆抗体制备及免疫胶体金银染色法 (IGSS)诊断的结果。用抗N .a的单抗结合间接免疫胶体金银染色法 (IGSS)对 6种微孢子虫进行检测 ,在光学显微镜下柞蚕微孢子呈现特异的褐色 ,能与包括家蚕微孢子虫在内的其它 5种微孢子虫加以区别。     

家蚕微孢子虫单克隆抗体的研制

以家蚕(Bombyx mori)微孢子虫(Nosema bombycis)免疫BALB/C小鼠的脾细胞与骨髓瘤细胞融合后制备杂交瘤细胞,分泌对N.bombycis孢子表面抗原特异性的单克隆抗体。用酶联吸附免疫分析(ELISA)以筛选杂交瘤细胞,经3次有限稀释法克隆后,用包括N.bombycis等6种微孢子虫及正常蚕体组织匀浆作抗原筛选出1株分泌仅对N.bombycis孢子有特异性单克隆抗体的杂交瘤细胞株(LC_2)。小鼠腹水滴度为1:640O,用ELISA最少检出100O个孢子的样本。     

安氏隐孢子虫COWP部分编码基因的表达及免疫原性

依据NCBI中安氏隐孢子虫囊壁蛋白(COWP)编码基因设计特异性引物,提取安氏隐孢子虫长春株总RNA,RT-PCR扩增目的基因AF,构建重组原核表达载体pET28a-AF,通过大肠杆菌BL21诱导表达,产物经SDS-PAGE以及Western blotting鉴定。然后纯化重组蛋白,通过免疫BALB/c小鼠进行体液免疫和细胞免疫水平的检测。结果显示,重组原核表达质粒pET28a-AF构建成功,Western blotting显示重组蛋白约为18 000,可被安氏隐孢子虫免疫小鼠的多克隆抗体和HRP标记的抗组氨酸抗体识别。重组蛋白免疫BALB/c小鼠的抗体水平差异显著(P0.05),与对照组相比,CD4+的值差异极显著(P0.01),而CD8+的值差异显著(P0.05)。结果表明,成功克隆并表达了重组安氏隐孢子虫囊壁蛋白,纯化的重组蛋白具有一定的免疫原性。     

新孢子虫NcGRA2t基因的原核表达及鉴定

为研究新孢子虫GRA2t基因的原核表达及其表达产物的免疫活性,用EcoRI和XhoI限制性内切酶从pMD20-NcGRA2t上切下NcGRA2t基因,克隆入pGEX-4T1中构建NcGRA2t-GST融合蛋白基因的重组表达载体pGEX-4T1-NcGRA2t。经测序验证后,在大肠杆菌BL-21(DE3)中诱导表达,表达产物采用谷胱甘肽-琼脂糖层析柱进行纯化。用Westernblotting方法,以犬感染新孢子虫Ncl株的血清鉴定表达产物的活性。结果,构建了pGEX-4T1-NcGRA2t原核表达质粒,并在大肠杆菌BL-21(DE3)中诱导表达分子量为46 ku的NcGRA2t-GST融合蛋白,表达产物经谷胱甘肽-琼脂糖层析柱纯化后,得到N端带有GST标签的NcGAR2t蛋白。Westernblotting结果显示,表达产物具有生物学活性。     

牛源犬新孢子虫NcSRS2-NcGRA7融合基因的克隆与生物信息学分析

为了解牛源犬新孢子虫NcSRS2-NcGRA7融合基因的生物学特性,本试验提取牛源犬新孢子虫基因组DNA,应用PCR技术扩增犬新孢子虫表面蛋白基因NcSRS2和致密颗粒蛋白基因NcGRA7,SOE-PCR技术拼接NcSRS2和NcGRA7基因,构建NcSRS2-NcGRA7融合基因重组克隆质粒,并进行PCR鉴定、双酶切鉴定及生物信息学分析。结果,NcSRS2基因扩增片段大小为1 061 bp,NcGRA7基因扩增片段大小为364 bp,NcSRS2-NcGRA7融合基因扩增片段大小为1 482 bp;获得重组克隆质粒pMD18-NcSRS2-NcGRA7经PCR鉴定、双酶切鉴定正确;测序分析表明,与Gen-Bank中已发表的美国株犬新孢子虫(AF061249、AF176649)核苷酸序列同源性为99%;经DNAman等软件分析,预测NcSRS2-NcGRA7融合蛋白抗原指数较高,融合蛋白二级结构以α-螺旋和β-折叠为主,三级结构中2种蛋白独立折叠,并借助Linker互相连接,功能互不影响。本试验为牛源犬新孢子虫NcSRS2-NcGRA7融合蛋白的免疫学研究奠定了基础。     

公羊犬肉孢子虫新种的发育

由犬传播的公羊犬肉孢子虫(Sarcoeystis arieticdnis sp.Nov)不同于羊犬肉孢子虫(S.ovicanis),也不同于早前报导的柔嫩肉孢子虫(S.tenela),並叙述了两个种的区别。试验中绵羊接种了3,000万个公羊犬肉孢子虫的孢子囊,在感染后19天第一代裂殖体即出现于肠系膜动脉与肠系膜淋巴结的动脉内。感染后的26—31天第二代裂殖体出现于各器官     

巨型住肉孢子虫烯醇酶基因克隆与表达

巨型住肉孢子虫(Sarcocystis gigantea)是羊体内一种常见寄生虫,多寄生于羊横纹肌内形成包囊,导致羊肉大量废弃,给畜牧业带来巨大经济损失。本研究尝试筛选能有效诊断住肉孢子虫感染的抗原。通过免疫印迹及质谱分析筛选到巨型住肉孢子虫候选诊断抗原烯醇酶,利用染色体步移技术扩增两侧翼未知序列并表达重组蛋白。应用生物信息学分析和免疫印迹对该蛋白诊断价值进行评价。结果筛选到的候选蛋白为巨型住肉孢子虫烯醇酶(SgENO),克隆得到1 181 bp的基因并获得重组蛋白rSgENO。对rSgENO应用进行初步评价,发现其与其他顶复亚门原虫的烯醇酶氨基酸相似性均较高(71%～92.1%),且能被弓形虫和新孢子虫阳性血清所识别,具有交叉反应性。本研究克隆并表达了巨型住肉孢子虫烯醇酶,后期评价发现该重组蛋白与新孢子虫和弓形虫有较强的交叉反应性,不能用于羊住肉孢子虫的血清学诊断,但有成为疫苗候选分子的潜力。     

Osmolarity- and Stage-Dependent Effects of Glycine on Parthenogenetic Development of Pig Oocytes

The osmolarities of media that are most effective for in vitro culture of mammalian oocytes and embryos are lower than that of oviductal fluid. Oocytes and embryos can survive the high physiological osmolarity in vivo perhaps owing to the presence of amino acids such as glycine, which serve as organic osmolytes in the female reproductive tract. In the present study, the effects of glycine on the parthenogenetic development of pig oocytes were examined in hypotonic or isotonic media. The results showed that culturing oocytes in isotonic media improved the cleavage rates (P<0.01) at 2 days in culture but inhibited any further development beyond cleavage when compared with the hypotonic media. However, addition of 4 mM glycine to the isotonic media resulted in improved blastocyst formation rates compared with that observed in the hypotonic media (P<0.01), and there was no inhibition of development beyond the cleavage stages in oocytes. The beneficial effects of glycine were observed only when oocytes were cultured in isotonic media and glycine was added at day 2 or 3 in culture. The results from the present study indicate that an isotonic medium with glycine is useful for in vitro culture of pig oocytes and that glycine may protect pig oocytes against the detrimental effects of increased osmolarity.     

Lesional expression of thymus and activation-regulated chemokine in canine atopic dermatitis

Glucocorticoids are reported to bias cytokines to a Th2 phenotype. However, this dogma has been advanced largely from studies utilizing potent glucocorticoid analogs. The current study was conducted to revisit the issue of glucocorticoid modulation of Th1/Th2 cytokine production and evaluate migration inhibitory factor (MIF) mRNA expression in cultured pig splenocytes treated with physiologically relevant concentrations of cortisol (CORT). Dexamethasone (DEX) was included for comparison. In Experiment 1, DEX, at 150 and 300 nM, suppressed concanavalin (ConA)-stimulated IFNgamma at both 12 and 24 h in culture, and IL-10 at 24h (P<0.05). Both 150 and 300 nM CORT suppressed IL-10 at 24 h (P<0.05), but neither concentration affected IFNgamma at 24 h. In Experiment 2, cells were cultured with a broader range of CORT for 48 h following ConA. Parallel cultures with identical treatments also were conducted in separate plates for evaluation of glucocorticoid regulation of MIF mRNA. IFNgamma was reduced by 300 nM DEX at 12, 24, and 48 h (P<0.05), whereas 150 and 300 nM CORT blunted IFNgamma at 24 h (P<0.05), but not 48 h. ConA increased IL-2 (P<0.01), but none of the steroid treatments affected IL-2. At both 12 and 24 h, IL-10 was reduced by 300 nM DEX and by 150 and 300 nM CORT (P<0.05). ConA increased relative abundance of MIF mRNA (P<0.001), but no steroid treatment affected MIF mRNA. In Experiment 3, steroid additions were delayed by 24 h after ConA, and cytokine concentrations evaluated 48 h later. Again, separate cultures were used for determination of effect of treatments on MIF mRNA. None of the steroid treatments affected IFNgamma, but 300 nM DEX reduced IL-10 (P<0.05). All of the CORT treatments (75-300 nM) reduced MIF mRNA (P<0.05), whereas DEX did not affect MIF mRNA in this experiment. The current experiments suggest that both DEX and high physiological concentrations of CORT can suppress both type 1 and type 2-like cytokines in cultured pig splenocytes. But, IL-10 was generally more sensitive to CORT suppression with increased time in culture than was IFNgamma. In addition, MIF mRNA could be suppressed by delayed addition of CORT to porcine splenocytes. Taken together, the data do not support the hypothesis that CORT directs the cytokine milieu toward a type 2 bias in cultured pig splenocytes.     

Serological response to pgp3 protein in animal and human chlamydial infections

Specific antibodies to plasmid-encoded protein pgp3 are known to be encountered in human Chlamydia (C.) trachomatis infections. In order to verify whether antibodies to this protein could be developed in animals infected with plasmid-carrying chlamydial strains, 454 animal sera were examined using a home-made pgp3 protein ELISA and Western blots (WB) of recombinant pgp3 protein from Chlamydophila (Cp.) psittaci. Likewise, 50 human sera were tested by ELISA and WB of recombinant pgp3 from C. trachomatis. The reactivity against pgp3 protein was compared to the reactivity against chlamydial elementary bodies (EBs) detected by microimmunofluorescence (MIF) test. The presence of pgp3-specific antibodies was demonstrated in most ducks and pigeons with Cp. psittaci infection detected by MIF, as well as in the majority of symptomatic cats and pigs infected with Cp. felis and C. suis, respectively, which reacted at high titres to Cp. felis and C. suis EBs by MIF. Moreover, most of the sera collected from patients with C. trachomatis culture-confirmed infection and seropositive to C. trachomatis by MIF, presented antibodies specific to C. trachomatis pgp3 recombinant protein. Therefore, pgp3 protein could be a useful marker of chlamydial infections in animals, as well as in humans.     

猪A组轮状病毒pW425et-Vp4基因工程乳酸菌的构建及表达

以嗜酸乳杆菌为受体菌株,将pW425et-Vp4重组质粒电转化入嗜酸乳杆菌,构建猪源A组轮状病毒Vp4基因工程乳酸菌,对影响电转化效率的主要因素进行研究,并对构建的乳酸菌进行表达分析.结果表明,在MRS培养基中加入1％甘氨酸+0.3 mol/L蔗糖,电场强度为11 kV/cm,脉冲时间为3.8 ms,电击后细胞复苏3～4 h等条件时,得到较高的转化效率,最高可达3.8×104 CFU/μg DNA. pW425et-Vp4乳酸菌阳性克隆经Western-blot分析表明,其蛋白具有与猪轮状病毒多克隆抗体的反应原性.     

定点突变的猪源性胰高血糖素样肽-2融合蛋白的原核表达与鉴定

为提高胰高血糖素样肽-2（GLP-2）生产效率以适应畜牧生产应用的需求,本试验利用基因工程技术表达出定点诱变的p[Gly2]GLP-2。用甘氨酸取代GLP-2氨基末端第2位的丙氨酸后,再根据大肠杆菌的偏好性对p[Gly2]GLP-2序列进行优化并在目标肽序列N-端添加肠激酶识别位点,合成基因序列,通过Kpn Ⅰ和Xho Ⅰ双酶切位点连接到pET-40b（+）构建原核重组表达载体p[Gly2]GLP-2-pET-40b（+）,重组质粒转化大肠杆菌BL21（DE3）感受态细胞,诱导表达重组蛋白。优化后最适表达条件为：菌液D_{600 nm}值为0.6时,用0.2 mmol/L IPTG在37℃诱导培养5 h;最终得到p[Gly2]GLP-2重组蛋白表达量较高的基因工程菌株。通过镍离子亲和层析柱纯化及分梯度洗脱获得纯度较高的p[Gly2]GLP-2融合蛋白,本结果为后续p[Gly2]GLP-2功能性的研究及其在畜牧生产的应用奠定了基础。     

Domain mapping and comparative binding features of eight dog IgE-specific reagents in ELISA, immunoblots, and immunohistochemistry.

Eight dog IgE-specific reagents including monoclonal and polyclonal antibodies (Ab) and a cross-reactive alpha chain of the human high affinity IgE receptor were mapped to recombinant fragments of the second (IgEf2) and third/fourth (IgEf3/4) domains of the dog IgE heavy chain. In ELISA, five out of eight reagents reacted to solid-phase bound IgEf2, of which two polyclonal Ab bound in addition to IgEf3/4. All Ab which recognized at least one recombinant IgE fragment, also bound to IgE in ELISA, immunoblots, and immunohistochemistry. In contrast, only one monoclonal Ab, that did not bind to the recombinant IgE fragments, reacted with immunoblots of serum and immunohistochemistry. The alpha chain could only be applied to ELISA with serum IgE. Furthermore, there was a wide range of heat-lability of binding reactions. Comparative analysis of available dog IgE-specific reagents enables more in-depth functional studies on IgE-mediated phenomena in dogs, and helps to further establish the dog as an animal model for allergy research.     

The effects of the Recombinant Mycobacterium Tuberculosis CFP10 Protein on the TLR Signal-mediated Inflammatory Responses in A549 Cells

The purpose of this study was to investigate the effects of recombinant 10 kDa culture filtrate protein (CFP10) of Mycobacterium tuberculosis on the inflammatory responses mediated by Toll-like receptor (TLR) signaling in A549 cells. The recombinant plasmid pCzn1-CFP10 was obtained by amplifying the cfp10 gene fragment using PCR and cloning it into the prokaryotic expression vector pCzn1. The obtained recombinant plasmid pCzn1-CFP10 was transformed into Escherichia coli BL21(DE3) and induced by IPTG to express the recombinant protein CFP10 (rCFP10). The purified rCFP10 protein was preserved after endotoxin was removed and desalted before use. The effect of rCFP10 treatment on the survival rate of A549 cells was detected by MTT assay. A549 cells were treated with rCFP10 to detect changes of key molecules of TLR signaling pathway and downstream inflammatory factors in A549 cells by qRT-PCR,Western blot and ELISA. The results showed that the recombinant expression vector pCzn1-CFP10 was successfully constructed and the high-purity rCFP10 protein was expressed and purified in this study. MTT assay showed that rCFP10 could inhibit the survival rate of A549 cells in a time- and concentration-dependent manner. In addition, rCFP10 could significantly (P<0.05) up-regulate the key molecules of TLR pathways TLR2, TLR4, MyD88, TRAF6, NF-κBp65 and downstream inflammatory cytokines IL-6 and TNF-α in A549 cells as compared to the control group. The recombinant Mycobacterium tuberculosis protein CFP10 could promote the secretion of cytokines by activating TLR receptor signaling pathway in A549 cells, which will provide a theoretical basis for further understanding of the pathogenesis of Mycobacterium tuberculosis.     

Growth hormone-binding protein in the goat: characterization, evolution under exogenous growth hormone treatment, and correlation with liver growth hormone receptor levels

This report describes the identification and characterization of a specific, high-affinity growth hormone-binding protein (GHBP) in lactating goat serum. Serum samples were incubated with [¹²⁵I]human GH as ligand and in the absence or in the presence of bovine GH as competitor. GH-GHBP complex formation was performed by high-performance liquid chromatography, and the radioactivity was recorded on-line with a Berthold LB detector connected to a computer. The results showed that a serum protein was able to bind specifically to human GH and bovine GH but not to ovine prolactin. Scatchard plots indicated an affinity constant of 4.5 × 10⁸ M⁻¹ and a maximum binding capacity of 4.8 × 10⁻¹⁰ mol/l. In addition, we conducted a 4-wk study to determine the effects of recombinant bovine GH administration on milk production in lactating goats. The effects of recombinant bovine GH treatment on milk production and on the regulation of GHBP and hepatic GH receptor levels were studied. As expected, recombinant bovine GH injected daily increased yields of milk, fat, protein (40, 61, and 40%, respectively), and circulating insulin-like growth factor 1 concentrations compared with controls. During the pretreatment and treatment periods, the control goats exhibited a constant amount of GHBP in serum. No consistent effect of GH treatment on GHBP level was observed. The binding of [¹²⁵I]bovine GH to hepatic microsomal membranes of GH-treated goats was significantly decreased compared with that of control goats. After MgCl₂ desaturation of membranes, the results demonstrated that the down-regulation of GH hepatic receptors, observed for the treated goat group, was induced by receptor occupancy without modification of binding affinity. The GH receptor gene expression, analyzed by slot blot and hybridization with an [-³²P]GH receptor cDNA probe, was not modified by the GH treatment. In lactating goats, the galactopoietic effect of exogenous GH involved a hepatic receptor occupancy. The individual concentration of GHBP in serum cannot explain the individual variations of responses to GH treatment in goats.     

绵羊肺腺瘤病毒受体透明质酸酶-2的原核表达及纯化

为建立绵羊肺腺瘤病毒(jaagsiekte sheep retrovirus,JSRV)受体透明质酸酶-2(Hyal-2)的原核高效表达体系,本试验设计了扩增JSRV受体Hyal-2基因的特异性引物,应用PCR技术扩增出Hyal-2全长基因,将该基因定向重组于原核表达载体pGEX-4T-1中,构建pGEX-4T-1-Hyal-2重组质粒,并转化到大肠杆菌BL21(DE3)中,经IPTG诱导表达,逐步优化条件至稳定表达,经Western blotting检测融合蛋白成功表达后,通过亲和层析法对融合蛋白进行纯化。结果表明,Hyal-2基因正确地插入到原核表达载体pGEX-4T-1中;经诱导含重组质粒pGEX-4T-1-Hyal-2的表达菌高效表达了带GST标签的目的蛋白;SDS-PAGE电泳结果显示目的蛋白分子质量为80 ku,与预期大小一致,经Western blotting验证为带GST标签的融合蛋白;通过谷胱甘肽亲和层析法获得纯化的目的蛋白。本试验结果为进一步制备Hyal-2蛋白的多克隆抗体及深入研究其功能奠定基础。     

蚕蛹壳聚糖甘氨酸衍生物的制备及其抗凝血作用研究

壳聚糖是生产抗凝血物质的理想原料。采用化学方法对蚕蛹壳聚糖进行分子修饰,即在蚕蛹壳聚糖分子上引入甘氨酸和环氧氯丙烷反应的中间体,制备壳聚糖甘氨酸衍生物。通过红外光谱及核磁共振分析其分子结构,结果显示甘氨酸已接枝到蚕蛹壳聚糖分子上,为在C6-OH发生醚化反应的壳聚糖甘氨酸衍生物。抗凝血试验结果表明,蚕蛹壳聚糖甘氨酸衍生物具有抗凝血活性,当其质量浓度达到2mg/mL时抗凝血效果最佳,可使血液完全不凝固。从凝血时间、凝血酶原时间、活化部分凝血活酶时间、凝血酶时间的检测结果可知,蚕蛹壳聚糖甘氨酸衍生物影响到外源性、内源性凝血系统和凝血活性酶的形成。依据抗凝血试验结果分析认为,蚕蛹壳聚糖甘氨酸衍生物中C6引入的羧基与壳聚糖C2上的氨基共同作用,产生了抗凝血效果。研究结果提示:蚕蛹壳聚糖甘氨酸衍生物可进一步开发为医用抗凝血材料。