共查询到19条相似文献，搜索用时 0 毫秒
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不同家蚕品种对家蚕核型多角体病毒(Bm NPV)的抵抗性存在较大差异,家蚕抗性品系AN、野BN对Bm NPV侵染具有较强的抵抗性。用易感家蚕品系C108分别与AN和野BN杂交、回交,构建两个抗性品系的BC3M和BC4M分离群体,利用简化基因组测序技术2b-RAD(基于IIB型限制性内切酶的RAD)进行亲本和杂交分离群体的基因组测序,通过生物信息学方法筛选位于染色体上与抗性相关的多态性SNP标记,进行抗性品种的基因型分析。对3个亲本及2个抗性分离群体样本基因组DNA构建的2b-RAD标签文库进行Illumina测序,获得抗性亲本AN、野BN的多态性SNP标记11 919个和12 293个。对抗性分离群体全基因组染色体的Bm NPV抗性关联SNP标记指数(SNP-index)进行分析,结果表明亲本品系AN、野BN的Bm NPV抗性相关SNP标记分布在多个染色体上,其中AN的全基因组中抗性贡献率前5位是第5、10、27、23和4号染色体,野BN的全基因组中抗性贡献率前5位的是第4、27、15、18和6号染色体,有多个SNP-index高值位点与已知Bm NPV抗性相关基因的位置具有较高的一致性。推测AN、野BN两个品系的Bm NPV高抗性与抗性主基因和其他抗性基因的联合作用有关。 相似文献
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《中国兽医杂志》2014,(12)
为了制备脑心肌炎病毒(EMCV)的单克隆抗体,并对其进行鉴定。应用杂交瘤细胞技术,将灭活EMCV免疫的小鼠脾细胞与骨髓瘤细胞融合,HAT选择培养、间接ELISA法筛选和多次克隆化,筛选出稳定分泌抗猪EMCV的杂交瘤细胞株,并对其分泌的单克隆抗体进行了亚型、效价、特异性和细胞株染色体核型的分析。结果获得了1株能分泌抗EMCV单克隆抗体的细胞株,命名为EMCV Y1G9,其分泌的抗体亚型为Ig G2b类;腹水效价可达3.2×104;细胞株的染色体核型分析结果是染色体众数为92±2,单抗的特异性也较好。制备出了具有良好特异性和敏感性的抗EMCV的Mc Ab,为建立EMCV的特异性检测方法及该病的防制奠定了基础。 相似文献
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为了探究t RNA修饰酶Mnm E对胸膜肺炎放线杆菌(APP)生长特性及致病性的影响，本研究以血清7型APP S8株DNA为模板经PCR分别扩增其Mnm E基因的保守结构域及全长基因，分别构建重组质粒pmnm E及pls88-Mnm E，并经PCR鉴定正确后将重组质粒pmnm E通过接合转移方式导入受体菌S8中，利用氯霉素抗性筛选Mnm E基因缺失株(S8△Mnm E)，并经PCR和测序鉴定；将重组质粒pls88-Mnm E电转化至S8△Mnm E感受态细胞中，经卡那抗性筛选Mnm E全长基因回补株(cS8△Mnm E)，并经PCR和测序鉴定。将上述两株菌分别在无抗性培养基中传10代每代均经相应PCR鉴定其遗传稳定性。采用q RT-PCR检测Mnm E基因突变对其上下游基因转录水平的影响。结果显示，突变株S8△Mnm E和回补株cS8△Mnm E均正确构建，且遗传稳定性均较好；q RT-PCR结果显示，S8△Mnm E株Mnm E上下游基因的转录水平均与亲本株无明显差异。通过检测不同时间点的OD600nm值并绘制生长曲线分析上述两种菌与亲本株分别在20种不同氨基酸单独缺失培养基中的生长... 相似文献
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《中国预防兽医学报》2018,(11)
为研究副猪嗜血杆菌(HPS)链霉素(SM)抗性的分子基础,本实验采用甲基磺酸乙酯(EMS)间断传代诱导HPS SC1401菌株,构建SM抗性突变株,测定其对SM的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),并对其耐药基因rpsL和rrs进行测序;通过对比分析筛选出rpsL基因中两个引起SM完全耐药的突变位点;为了进一步验证突变位点与抗性的关联性,设计位点突变引物以构建突变型质粒,并分别转化到亲本株中,获得单一位点突变菌株,测定其对SM的MIC和MBC。结果显示:EMS诱导的SM抗性突变菌株rpsL基因第43号(AAA→AGA)或第88号密码子(AAA→AGA)发生了突变,rrs基因未检测到突变;该抗性突变菌株与单一位点突变菌株1401D43和1401D88对SM的MIC和MBC均大于8 192μg/mL,远高于亲本株SC1401的SM抗性水平。实验结果表明,HPS的SM抗性主要由rpsL基因特定位点突变引起,其中,rpsL基因第43号和第88号密码子为主要突变位点。本实验为完善HPS耐药分子机制研究,特别是对氨基糖苷类药物产生耐药的分子基础提供一定的参考。 相似文献
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通过构建标记疫苗株来解决流产布氏杆菌(B.abortus)鉴别诊断方面的缺陷,本研究以bp26基因作为重组靶住点,S19为亲本,利用bp26基因ORF外侧序列作为同源重组序列,卡那霉素抗性基因(Kanr)为抗性筛选标记,通过双交叉重组筛选获得bp26基因缺失突变的重组S19株,命名为S19-△26.小鼠感染结果表明,突变株S19-△26的残留毒力与亲本株S19相比较没有发生明显改变,康复时间约为15周,突变株S19-△26、亲本株S19和B.abortus强毒株S544接种小鼠后的第3周能检测出"O"抗原的特异性抗体,而第6周开始S19和S544接种小鼠BP26特异性抗体明显升高,S19-△26接种的小鼠一直没检测到BP26特异性抗体.小鼠免疫保护试验显示,脾脏分离CFU数比空白对照要低310g10,S544攻击后脾脏细菌分离数表明突变株具有与亲本疫苗株免疫保护性无明显差异.结果表明,S19-△26免疫能够通过血清学方法与野生型B.abortus感染后的免疫反应相区别,具备作为标记疫苗的潜力. 相似文献
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重组转移载体pBSKA通过电转化导入亲本菌胸膜肺炎放线杆菌血清7型（APP-7）WF83株,电转化后的产物涂布于TSB／Kan平板,2d获得突变株。卡那霉素抗性实验证实突变株有卡那霉素抗性;NAD依赖性实验证实突变株依赖NAD生长;PCR鉴定证实了卡那霉素抗性基因置换了apxlIC基因,并证实突变株中无pBSKA质粒的存在;溶血活性实验证实突变株完全失去了溶血活性;细胞毒性实验证实突变株的细胞毒性完全丧失;对小鼠的安全性实验证实突变株的毒力显著减弱,突变株对小鼠是安全的;遗传稳定性实验证实,突变株在体外连续传30代和在体内传10代均不会发生卡那霉素抗性的丢失。结果表明实验成功构建了基因缺失减毒株,为进一步以此突变株作为基因工程弱毒活疫苗株开展研究奠定了一定的基础。 相似文献
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为研究Hfq蛋白在支气管败血波氏杆菌(Bb)的致病作用,本研究采用同源重组技术,将卡那霉素抗性基因替换Bb122株的hfq基因,构建BbΔhfq突变株。将其连续传20代,Δhfq突变株具有良好的遗传稳定性。生化检测试验表明,突变株与亲本株未发生明显改变。此外,在37℃培养时,突变株与亲本株生长曲线无明显差异,但在42℃培养时,突变株的生长曲线略低于亲本株。抗逆性试验表明,突变株与亲本株在紫外线照射和50℃条件下,亲本株的耐受能力均显著高于突变株(p0.05)。然而,将亲本株与突变株分别以2×109cfu/只腹腔接种小鼠时,亲本株接种小鼠全部死亡,而突变株接种小鼠则全部存活,表明Hfq蛋白与该菌的致病性有关。本研究为进一步研究Hfq蛋白的功能及其在Bb致病机制中的作用奠定了基础。 相似文献
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参照布氏锥虫次黄嘌呤鸟嘌呤磷酸核糖转移酶 ( HGPRT)基因的核苷酸序列 ,设计合成了 1对引物 ,引物间距为 63 0 bp,包含完整的 HGPRT基因。以伊氏锥虫湖北水牛株总 RNA为模板进行 RT-PCR扩增 ,琼脂糖凝胶电泳显示 ,获得 1条长约 63 0 bp的特异性条带 ,符合设计要求。扩增片段克隆于 p GEM-T Easy载体 ,经筛选鉴定 ,证明已获得了 HGPRT基因阳性克隆。核苷酸序列分析表明 ,克隆的 HGPRT基因在重组质粒中的连接向位和阅读框架是正确的。二级结构和疏水性分析表明 ,HGPRT基因产物具有复杂的空间结构 ,提示有良好的抗原性 相似文献
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Bovine B-cell hybrids were generated by fusing pokeweed mitogen (PWM) activated normal bovine peripheral blood lymphocytes (PBL) with an aminopterin-sensitive bovine B-cell line BL20. The fusion partner was derived by irradiation and growth in medium containing increasing concentrations of 6-thioguanine (6TG). Resultant cells were designated ATS/BL20. Polyethylene glycol-induced (PEG) fusion was used and hybrids were selected in hypoxanthine-aminopterin-thymidine (HAT) medium. Hybrid cell growth was noticed after 4 to 6 weeks of fusion following a period of quiescence. Hybrid formation was confirmed by selection in HAT medium, expression of cytoplasmic IgM (cIgM) and surface antigens and karyotype analysis. 相似文献
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制备配对胃蛋白酶原Ⅱ(PGⅡ)单抗,建立人血清中胃蛋白酶原Ⅱ夹心ELISA检测方法。用胃蛋白酶原Ⅱ免疫Balb/c小鼠,制备免疫脾细胞,与SP2/0融合,用HAT培养基进行筛选培养,间接ELISA检测阳性克隆,对阳性孔进行多次单克隆化,选出效价高、分泌性能稳定的杂交瘤细胞,制备腹水并进行纯化。进行单抗配对,建立胃蛋白酶原Ⅱ双抗体夹心检测方法。获得1D8、1C6、2H11、2E3等4株杂交瘤,经配对试验,确定1D8、2H11可作为夹心ELISA检测PGⅡ的单抗。成功制备出配对单抗,初步建立了PGⅡ双抗体夹心ELISA方法。 相似文献
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用RPMI1640培养基对抗牛布鲁氏菌独特型抗体杂交瘤细胞株(F9)进行培养，并对其生长代谢进行测定。该细胞不论接种1.1×10 相似文献
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Shin-Ichi Tanabe Laetitia Bonifait Nahuel Fittipaldi Louis Grignon Marcelo Gottschalk Daniel Grenier 《Canadian journal of veterinary research》2010,74(1):65-70
In this study, an unencapsulated Streptococcus suis mutant was used to investigate the pleiotropic effects resulting from capsule loss. The capsule deficient mutant of S. suis acquired a biofilm-positive phenotype, which was associated with significantly increased cell surface hydrophobicity. Cell-associated fibrinogen-binding and chymotrypsin-like activities were decreased in the unencapsulated mutant. The mutant did not differ significantly from the encapsulated parent strain for minimal inhibitory concentrations to penicillin G, ampicillin, and tetracycline. However, while the encapsulated strain was highly resistant to the bactericidal action of penicillin G and ampicillin, the unencapsulated mutant was approximately 60-fold more sensitive. Compared with the parent strain, the unencapsulated mutant induced a much higher inflammatory response in monocyte-derived macrophages resulting in an increased secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8. The capsule appears to hinder important adhesins or hydrophobic molecules that mediate biofilm formation, as well as cell wall components capable of stimulating immune cells. 相似文献
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ZHANG Wei-ran DAI Yang WANG Yi-min LIU Xin-feng WANG Ting GUO Hong DING Xiang-bin 《中国畜牧兽医》2015,42(7):1763-1769
To investigate the effect of basic fibroblast growth factor (bFGF) on the proliferation of bovine skeletal muscle satellite cells,bovine skeletal muscle satellite cells were isolated and cultured in the medium with bFGF,the growth and differentiation state of muscle satellite cells were observed,and the growth curve and EDU cell proliferation assay were conducted.The results showed that cell morphology of bovine skeletal muscle satellite cells cultured in medium containing bFGF was better and proliferation rate was significantly higher than that in control group (P<0.05).The results indicated that bFGF could promote proliferation of bovine skeletal muscle satellite cells efficiently in growth medium and differential medium.Our study established an efficient method to culture bovine skeletal muscle satellite cells,which could provide reference for studying and using of skeletal muscle satellite cells. 相似文献
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为研究碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对牛骨骼肌卫星细胞增殖的影响,试验对牛骨骼肌卫星细胞进行体外分离培养,在生长培养基和分化培养基中分别添加bFGF,观察细胞生长及分化情况,绘制细胞生长曲线,并进行EDU细胞增殖检测试验。结果显示,添加了bFGF培养基的细胞生长状态较对照组良好,细胞增殖速度快,细胞增殖率显著高于对照组(P<0.05),说明bFGF对牛骨骼肌卫星细胞在生长培养基和分化培养基中增殖都具有良好的促进作用。本研究建立了一种高效的牛骨骼肌卫星细胞培养方案,为骨骼肌卫星细胞的研究利用提供参考。 相似文献
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采用脂质体法将具有抑制猪繁殖与呼吸综合征病毒(PRRSV)复制的shRNA质粒pEGFP-N1-shRNA导入PK-15细胞中,经G418药物筛选后,分离扩增绿色荧光蛋白阳性细胞,获得抗PRRSV转基因PK细胞系。通过对转染方法和条件的优化,确立最佳的转染及筛选步骤;对得到的阳性转基因细胞进行冷冻-解冻,并作PCR检测。结果表明,G418最佳筛选浓度为600μg/mL,脂质体与质粒的最佳转染比例为7∶2,最佳转染时间为24 h。本研究成功建立抗PRRSV转基因细胞系,为进一步的功能验证及体细胞核移植奠定了基础。 相似文献
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New multidrug resistant cell lines developed from the canine B cell lymphoma cell line (GL-1) were characterized in terms of chemosensitivity to some antineoplastics and P-glycoprotein (Pgp) expression. GL-1 was continuously exposed to a culture medium containing gradually increasing levels of doxorubicin and the cells that could grow in the presence of doxorubicin were obtained. Chemosensitivity of these cells to various antineoplastics were investigated with or without verapamil, which reversed Pgp-mediated drug resistance. The expression of Pgp on the cells was also examined by Western blot analysis. As a result, three kinds of resistant cell lines, designated as GL-DOX60, 300, and 4000 were obtained. These cell lines showed stable proliferation in the medium containing 60, 300, and 4000 ng/ml, respectively. These cells were much more resistant to vincristine than doxorubicin. This resistance was strongly reversed by the presence of verapamil. On the other hand, cisplatin was effective enough in killing these derived cells. In the Western Blot analysis, some bands that reacted to the anti-human Pgp monoclonal antibodies were observed in GL-DOX4000. The cells derived from GL-1 have multidrug resistance potential mediated by canine Pgp. The cells produced in this experimental trial are considered to be useful models for various investigations on canine multidrug resistance. 相似文献