奶牛粒细胞-巨噬细胞集落刺激因子的原核表达及其活性检测

为了表达奶牛粒细胞-巨噬细胞集落刺激因子(GM-CSF)并对其活性进行检测.根据GenBank中登录的牛GM-CSF基因序列(U22385),设计一对特异性引物;采用RT-PCR方法,以LPS体外诱导的奶牛肺泡巨噬细胞为材料,从总RNA中扩增出奶牛GM-CSF cDNA基因,克隆到pGEM-T载体中,经酶切鉴定与序列测定,结果显示克隆的奶牛GM-CSF基因与GenBank中登录的牛GM-CSF基因序列的核苷酸和氨基酸的同源性分别为99.7%和99.3%.构建pET32a-GM-CSF原核表达重组质粒,经IPTG诱导表达,SDS-PAGE结果显示重组蛋白大小约为35 ku.分别运用集落形成试验与MTT比色法测定重组蛋白活性,结果显示重组奶牛GM-CSF蛋白能够诱导粒细胞前体呈集落性生长并具有较强的体外增殖淋巴细胞的活性,为下一步临床试验奠定了基础.     

Protective immune responses induced by in ovo immunization with recombinant adenoviruses expressing spike (S1) glycoprotein of infectious bronchitis virus fused/co-administered with granulocyte-macrophage colony stimulating factor

Infectious bronchitis virus (IBV) causes tremendous economic losses associated with production inefficiencies and mortality in poultry industry worldwide. In the present report, the recombinant adenoviruses expressing chicken granulocyte-macrophage colony stimulating factor (GM-CSF) and S1 gene of nephropathogenic IBV were constructed and characterized. Then, the immunological efficacy and protection against homologous IBV challenge were assessed in specific pathogen free (SPF) chickens. The results showed that the chickens vaccinated in ovo with rAd-S1, rAd-GM-S1 (GM-CSF fused with S1 using glycine linkers) and rAd-GM-CSF plus rAd-S1 (co-administered) developed specific anti-IBV HI antibodies. Moreover, the fusion of the GM-CSF markedly increased spleen cell proliferation and IFN-γ production while mild increased in IL-4 production, which demonstrated the enhancement of cell-mediated immune responses. Following challenge with IBV, the chickens in the group vaccinated with rAd-S1 fused or co-administered with GM-CSF had fewer nephropathic lesions and showed 100% protection as compared to that of rAd-S1 alone which showed 70% protection. It indicated that the single dose in ovo vaccination of the GM-CSF fused or co-administered with S1 of IBV could enhance significantly the humoral, cellular immune responses and provide complete protection against nephropathogenic IBV challenge. This finding may provide basic information for effective in ovo vaccines design against IBV.     

干扰素-τ(IFN-τ)的分子结构和妊娠识别机理

在反刍动物的整个妊娠失败中，早期胚胎死亡占有很大的比例，它在很大程度上是由母体妊娠识别失败造成的IFN-τ（干扰素-τ）是由反刍动物孕体附植时发出的特有的妊娠识别信号，它是建立母体妊娠识别的一种主要因子，本文阐述了IFN-τ的分子结构及其最新研究进展，即IFN-τ和PGF2α，UCRP，OTR，GM-CSF的互作机理。     

The Effects of Growth Factor on Testicular Germ Cell Apoptosis in the Stallion

Apoptosis is necessary for both initiation and control of spermatogenesis; however, an increase in apoptosis can lead to subfertility/infertility in stallions, causing substantial financial loss in the equine industry. The ability of stem cell factor (SCF), leukemia-inhibiting factor (LIF), granulocyte–macrophage colony-stimulating factor (GM-CSF), and estradiol (E₂), alone or in combination, to prevent apoptosis of germ cells in short-term equine testicular cultures was examined. Testicular tissue was sectioned into approximately 2-mm cubes and placed in media-filled culture chambers. Concentrations of SCF (100 ng/mL), LIF (10 ng/mL), GM-CSF (5 ng/mL), and E₂ (10⁻⁹ mol/l) were added alone or in combination to each well. After 6 hours in culture, the tissue was fixed and immunohistochemically (terminal deoxynucleotidyl transferase-mediated nick-end labeling; TUNEL) stained for apoptosis detection. Apoptotic cells per 100 Sertoli cell nuclei within seminiferous tubules were counted until the 500^th Sertoli cell nuclei was reached. This counting procedure was used for each slide. An analysis of variance (ANOVA) with a Tukey's test was used to compare apoptotic rates. In comparison with the control, GM-CSF alone lowered apoptosis by 34.77%. GM-CSF–treated tissue combined with SCF and LIF as well as GM-CSF combined with SCF, LIF, and E₂ reduced apoptosis when compared with the control (37.45% and 44.40%, respectively) or other treatment combinations. GM-CSF alone reduced apoptosis; results suggest possible synergy for the combinations of SCF and LIF with GM-CSF and for E₂ with SCF, LIF, and GM-CSF.     

巨噬细胞集落刺激因子(GM-CSF)对小鼠胚胎体外发育的影响

以小鼠自然受精与体外受精胚胎为模型,探索鼠源巨噬细胞集落刺激因子(GM-CSF)在小鼠胚胎早期发育过程中的生理功能。分别使用添加不同剂量(对照组:0 ng.mL-1;试验组:0.5、2和10 ng.mL-1)GM-CSF的化学限定培养基连续培养小鼠自然受精、体外受精原核期与2细胞期胚胎,检测其胚胎着床前发育效率(卵裂率、囊胚率)及囊胚的质量(囊胚总细胞数、ICM/总细胞数的比率、凋亡指数)。结果发现,对自然受精胚胎而言,原核时期添加不同剂量的GM-CSF,其卵裂率、囊胚总细胞数、ICM/总细胞数的比率在各组之间均差异不显著,但10 ng.mL-1试验组的囊胚率显著低于对照组(61.6±5.1)%(P0.05);2细胞时期添加不同剂量的GM-CSF,其囊胚率、囊胚总细胞数、ICM/总细胞数的比率在各组之间均差异不显著,但试验组中的囊胚凋亡指数均显著低于对照组(P0.05)),并且试验组中2 ng.mL-1的囊胚凋亡指数最低,显著低于0.5 ng.mL-1(P0.05),其他试验组之间的囊胚凋亡指数没有统计学上的差异性。对体外受精胚胎来说,原核时期添加GM-CSF,其卵裂率、囊胚率、囊胚总细胞数、ICM/总细胞数的比率在各组之间均无显著性差异;在2细胞时期,10 ng.mL-1组的囊胚率显著低于对照组及其他2个试验组(P0.05),但各组之间的囊胚总细胞数、ICM/总细胞数的比率均无显著性差异。本研究结果表明,在化学限定培养基中添加一定剂量的GM-CSF有利于改善小鼠自然受精与体外受精囊胚的质量,但剂量过高可能不利于小鼠受精胚胎的早期发育。     

共表达PCV2衣壳蛋白和猪GM-CSF重组质粒的构建与体外表达

将猪圆环病毒2型(Porcine circovirus type 2, PCV2)orf2和猪粒细胞-巨噬细胞集落刺激因子(gm-csf)基因插入同一哺乳动物细胞表达载体pIRESneo中，构建出重组共表达质粒pIRES-orf2/gm-csf。以脂质体转染方法将重组质粒pIRES-orf2/gm-csf转染COS7细胞，经间接免疫荧光实验和集落刺激形成实验检测，结果表明orf2和gm-csf基因在COS7细胞中得到表达，转染细胞呈现特异性荧光，转染细胞培养上清能剌激猪骨髓细胞形成集落。重组质粒pIRES-orf2/gm-csf的成功构建为进一步研究猪圆环病毒2型的DNA疫苗奠定了基础。     

小鼠对pGM-CSF/SS生长抑素基因疫苗的免疫应答

通过研究细胞因子GM-CSF与生长抑素pcS/2SS融合表达质粒pGM-CSF/SS诱导小鼠免疫应答的影响,探求生长抑素基因免疫新策略。将60只20日龄雄性昆明小白鼠均分为6组,分别用PBS(C0)、pGM-CSF质粒(C1)、pGM-CSF/SS质粒(T1、T2、T3)、pcS/2SS(T4)质粒口服免疫,T1~T4组的免疫剂量分别为2×106cfu/mL、2×108cfu/mL、2×1010cfu/mL、2×108cfu/mL,以上质粒均以减毒沙门氏菌为传导载体。结果显示:pGM-CSF/SS组的抗生长抑素抗体AP/AN值显著高于pcS/2SS组,阳性鼠体增重亦高于pcS/2SS组,抗体阳性率分别是71.4%和40.0%。另外,高剂量组的AP/AN值极显著高于低剂量组,显著高于中剂量组;中剂量组的AP/AN值显著高于低剂量组,高剂量组的抗体阳性率也明显高于中剂量组和低剂量组,分别为100%、71.4%和20.0%。可见,pGM-CSF/SS疫苗的免疫效果优于pcS/2SS疫苗,且pGM-CSF/SS疫苗呈剂量依赖关系。这些结果显示出细胞因子GM-CSF可以增强生长抑素DNA疫苗的免疫效果。     

鸡粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因的克隆及生物信息学分析

为了解GM-CSF的基因及蛋白质特性,本研究以pUC-GM-CSF为模板,根据GenBank中登录的GM-CSF序列设计引物扩增GM-CSF基因,随后将其克隆至 pMD18-T载体,并对鉴定正确的重组质粒 pT-GM-CSF进行测序分析,然后对GM-CSF基因进行同源性分析、ORF分析、氨基酸序列分析、疏水性分析、磷酸化位点预测、亚细胞定位分析及二级结构预测。结果表明：该基因ORF的全部长度为453 bp,能够编码150个氨基酸,并且在GM-CSF基因所编码的蛋白质中有4个丝氨酸和3个苏氨酸可能成为蛋白激酶磷酸化的位点。同时,GM-CSF蛋白的最大疏水性为2.267,且有8.91%的可能位于细胞外,有0.8%的可能位于细胞壁,有0.24%的可能位于细胞质。同时,该蛋白质的二级结构主要由无规卷曲、α螺旋和延伸链构成。本研究通过对GM-CSF基因进行生物信息学分析,为探究其免疫增强机制奠定基础。     

以减毒沙门氏菌为载体的GM-CSF与生长抑素DNA疫苗的安全性和稳定性

将含GM-CSF基因与生长抑素(SS)的融合真核表达质粒pGM-CSF/SS转入减毒沙门氏菌株CS022。选择40只22~24 g的雌性小鼠,随机分成4组,其中1组为对照组,口服生理盐水;2~4组为试验组,分别用上述重组减毒沙门氏菌株CS022以108、109、1010CFU的剂量口服免疫小鼠,2周后以相同的剂量加强免疫,研究pGM-CSF/SS真核表达质粒在体内的稳定性和减毒沙门氏菌对小鼠的安全性。同时通过体外传代,研究该真核表达质粒在体外的稳定性。结果表明:108、109CFU剂量口服小鼠,成活率达100%,而1010CFU剂量口服小鼠成活率降低至90%,腹泻率达50%。体外传10代和口服免疫4周后从小鼠肝脏和脾脏分离的减毒菌用琼脂糖凝胶电泳和酶切鉴定法证实,减毒菌中的重组质粒在体外和侵入小鼠体内过程中具有较好的稳定性。质粒DNA在传10代以后,虽然每代的质粒DNA含量有些变化,但总体趋势基本是恒定的,第10代时,质粒DNA的含量与第1代的含量差异不显著。上述结果提示,利用该减毒沙门菌作为载体传递pGM-CSF/SS DNA疫苗免疫小鼠具有相对安全性和稳定性,为进一步有效激发机体的免疫应答提供了前提。     

羊传染性脓疱病毒疫苗株和野毒株GIF基因比较分析

羊传染性脓疱(orf)是由羊传染性脓疱病毒(orf virus,ORFV)感染引起的绵羊和山羊的一种接触性人兽共患病。粒细胞-巨噬细胞集落刺激因子(GM-CSF)/白细胞介素(IL-2)抑制因子(GIF)基因为ORFV编码中晚期病毒基因,它能够与GM-CSF、IL-2结合并且抑制二者的活性,从而抑制宿主抗病毒作用。为了比较疫苗毒株和流行毒株间GIF的差异,本试验扩增并测定了ORFV疫苗株和野毒株的GIF基因序列,比较分析了ORFV疫苗弱毒株和野毒株GIF基因的核酸水平和氨基酸水平的差异情况,以及二、三级蛋白结构。结果表明:本次测定的ORFV疫苗株与野毒GIF基因核苷酸序列相似性为94.3%,氨基酸序列相似性为92.5%,两者的蛋白三级结构预测上也有差异。本研究结果为该病的防控提供了参考依据。