猪FcγRIII的原核表达及多克隆抗体的制备

目的：构建猪FcγRIII 基因的原核表达载体,诱导表达重组蛋白,制备鼠抗猪 FcγRIII 抗血清。方法：从质粒pTG19-T-FcγRIII中用PCR方法克隆到编码完整猪FcγRIII蛋白分子的基因片段,将其插入到原核表达载体pET-32a中,构建了猪FcγRIII 原核表达载体pET-FcγRIII ,转化大肠杆菌BL21 (DE3) ,IPTG诱导蛋白表达,经尿素洗涤纯化后,以纯化后的融合蛋白FcγRIII-His 为抗原免疫小鼠,获得抗血清。Western blotting、ELISA 法鉴定获得的抗血清,ELISA 结果显示抗体效价为1∶16000,具有高度特异性,免疫印迹结果显示制备的多抗可以与重组猪FcγRIII蛋白特异性结合。结果：成功构建猪FcγRIII原核表达载体,纯化到融合蛋白FcγRIII-His,用纯化的融合蛋白免疫小鼠制备了多克隆抗体,Western blotting、ELISA 法证实多克隆抗体制备成功。结论：成功获得了猪 FcγRIII 多克隆抗体,为进一步研究猪FcγRIII 蛋白的功能奠定了基础。     

猪FcγRⅢ的原核表达及多克隆抗体的制备

目的:构建猪FcγRIII基因的原核表达载体,诱导表达重组蛋白,制备鼠抗猪FcγRIII抗血清。方法:从质粒pTG19-T-FcγRIII中用PCR方法克隆到编码完整猪FcγRIII蛋白分子的基因片段,将其插入到原核表达载体pET-32a中,构建了猪FcγRIII原核表达载体pET-FcγRIII,转化大肠杆菌BL21(DE3),IPTG诱导蛋白表达,经尿素洗涤纯化后,以纯化后的融合蛋白FcγRIII-His为抗原免疫小鼠,获得抗血清。Western blotting、ELISA法鉴定获得的抗血清,ELISA结果显示抗体效价为1∶16000,具有高度特异性,免疫印迹结果显示制备的多抗可以与重组猪FcγRIII蛋白特异性结合。结果:成功构建猪FcγRIII原核表达载体,纯化到融合蛋白FcγRIII-His,用纯化的融合蛋白免疫小鼠制备了多克隆抗体,Western blotting、ELISA法证实多克隆抗体制备成功。结论:成功获得了猪FcγRIII多克隆抗体,为进一步研究猪FcγRIII蛋白的功能奠定了基础。     

糖原合成酶激酶3在猪气道上皮细胞鳞状分化中作用的初步探讨

为探讨糖原合成酶激酶3（glycogen synthase kinase 3,GSK3）在气道（气管和支气管）上皮细胞鳞状分化中的作用,培养原代猪气道上皮细胞,用GSK3的高度选择性抑制剂氯化锂处理,观察细胞形态变化,用Western blot检测β-连环素、磷酸化GSK3和鳞状分化标记物外皮蛋白的表达、RT-PCR检测鳞状分化标记物小脯氨酸丰富蛋白mRNA的表达、荧光素酶报告基因分析β-连环素／Tcf信号的激活状态。结果显示,锂能诱导猪气道上皮细胞出现鳞状形态、增加小脯氨酸丰富蛋白mRNA和外皮蛋白的表达、促进GSK3的抑制性丝氨酸磷酸化和β-连环素的细胞核内转位;锂能激活β-连环素／Tcf信号,但该作用出现于鳞状分化标记物增加之后。上述结果提示,GSK3可能参与猪气道上皮细胞的鳞状分化。     

猪源戊型肝炎病毒基因IV型ORF3编码蛋白的表达及鉴定

通过PCR从已构建的猪源戊型肝炎病毒全基因克隆扩增ORF3全基因,将扩增产物插入到pMD18-T载体中,亚克隆至原核表达载体pET28a（+）,构建pET28a-ORF3表达载体,转入E.coli BL21 (DE3),IPTG诱导表达。Ni-NTA层析柱纯化表达蛋白,用SDS-PAGE、免疫印迹、ELISA等方法分析鉴定表达产物。结果成功扩增到345 bp的目的基因;构建了重组表达载体pET28a-ORF3;转化宿主菌E.coli BL21 (DE3)后表达产物的相对分子质量在6.50~16.5 kDa之间,与预期表达的目的蛋白相对分子质量相符;表达的目的蛋白能与阳性猪源和人源血清发生特异性反应,证实其具有较好的反应原性。     

反转录病毒载体介导的猪APOBEC3F在猪源细胞PK15中的表达

目的:利用反转录病毒载体构建猪载脂蛋白B mRNA编辑酶催化多肽样蛋白(APOBEC)3F重组质粒,并实现其在猪肾细胞PK15中的表达。方法:用RT-PCR方法扩增五指山猪来源的外周血淋巴细胞APOBEC3F基因,将其定点插入反转录病毒载体pMSCV neo中,同时于插入位点两侧分别添加FLAG和GFP标签,构建重组质粒pMSCV-FLAG-A3F-GFP,并进行酶切、测序鉴定;将鉴定正确的重组质粒与pVSV-G、pGag-Pol共转染包装细胞HEK293T,分别于转染后48~72 h收集细胞的培养上清以获得假型病毒粒子;用该假型病毒感染猪源细胞PK15,通过PCR、Western印迹检测目的基因的整合及表达。结果:PCR扩增到1254 bp的猪APOBEC3F基因,重组质粒pMSCV-FLAG-A3F-GFP经酶切、测序,结果无误;3质粒共转染HEK293T细胞包装出的假型病毒感染PK15细胞后观察到GFP表达;从感染假型病毒的PK15细胞基因组中扩增到1254 bp的猪APOBEC3F基因,Western印迹检测到78.1×103的猪APOBEC3F蛋白的表达。结论:实现了反转录病毒载体介导的猪APOBEC3F在猪源细胞PK15中的整合与表达,为深入研究该分子对猪内源性反转录病毒(PERV)的抑制作用奠定了基础。     

METTL3对猪干细胞多能基因表达的调控作用

m~6A是真核生物m RNA中重要的转录后修饰,METTL3作为m~6A甲基转移酶复合物中的重要组分,在细胞重编程、胚胎干细胞和诱导多能干细胞的干性维持、胚胎发育等过程中发挥重要作用。为了揭示猪METTL3的表达模式,对不同物种METTL3蛋白序列进行了比对,用RT-PCR检测了METTL3基因在不同猪组织和细胞中的表达情况,并确认了METTL3的细胞核定位。为了研究METTL3对猪干细胞多能基因表达的调控作用,克隆了猪METTL3编码区序列,设计了METTL3干扰片段,并构建了相应的过表达和沉默载体。发现干扰METTL3的表达后,猪多能干细胞出现类似na?ve状态的细胞克隆,NANOG、OCT4和LIN28A表达水平显著升高。在猪多能干细胞培养基中添加m~6A甲基化抑制剂环亮氨酸培养细胞48 h后,试验结果与干扰METTL3表达的结果一致。本研究为优化猪多能干细胞的培养体系提供了新的方向和依据。     

猪α-干扰素的原核表达及活性测定

目的：表达并纯化出具有生物学活性的重组猪α-干扰素。方法：根据前期合成的猪α-干扰素基因序列设计引物,用PCR方法扩增出猪α-干扰素基因,并将其定向克隆入原核表达载体pET28中,酶切及测序鉴定正确后,转化大肠杆菌BL21进行诱导表达,对表达产物通过镍琼脂糖凝胶柱亲和层析纯化、透析法复性后,采用微量细胞病变抑制法测定重组猪α-干扰素的活性。结果：测序结果表明构建了猪α-干扰素原核表达载体pET28-poIFN-α;诱导表达后经SDS-PAGE分析,在相对分子质量约21×10^3的位置出现明显的诱导蛋白条带,与目的蛋白大小相近;经分析表达产物主要以包涵体形式存在,约占菌体总蛋白的36%,纯化后的目的蛋白纯度约为90%;采用微量细胞病变抑制法测定其活性约为1.67×10^6U/mg。结论：获得了纯度较高并具有较高活性的重组猪α-干扰素,为下一步研究猪α-干扰素药物价值及其生物制剂的生产、应用奠定了基础。     

沉默Gpr3基因表达对猪卵泡颗粒细胞凋亡的影响

G蛋白偶联受体3（G protein-coupled receptor 3,Gpr3）属于G蛋白偶联受体超家族成员,能够维持卵泡卵母细胞减数分裂的前期阻滞,但在卵泡颗粒细胞中的作用不清。该研究利用RNAi技术,以化学合成的siRNA转染体外培养的猪卵泡颗粒细胞,并利用Real-time PCR和Western blot技术检验Gpr3基因的沉默效果;利用MTT（四甲基偶氮唑盐）、流式细胞术和Real-time PCR技术检测沉默Gpr3基因表达对猪卵泡颗粒细胞凋亡以及凋亡相关基因表达的影响。结果显示,Gpr3-siRNA能够有效地抑制猪卵泡颗粒细胞中Gpr3基因mRNA和蛋白的表达（P〈0.01）;在沉默Gpr3基因表达后,猪卵泡颗粒细胞的细胞活性由0.419升高至0.586,同时细胞凋亡率由2.67%下降至0.42%,并在显著上调Bcl-2表达的同时,下调了Bax的表达（P〈0.05）。结果表明,沉默Gpr3基因的表达抑制了猪卵泡颗粒细胞的凋亡,其机制可能与调控Bcl-2和Bax表达有关。     

过表达Gpr3诱导猪颗粒细胞G1阻滞及凋亡

G蛋白偶联受体3（Gpr3）属于G蛋白偶联受体视紫质家族成员. Gpr3通过激活Gs蛋白介导的下游信号通路,维持卵泡卵母细胞减数分裂的前期阻滞,但在卵泡颗粒细胞中的作用不清. 为了明确Gpr3在猪卵泡颗粒细胞中的功能,构建了Gpr3基因的真核表达载体,利用过表达的方式激活其介导的信号通路,并利用MTT、流式细胞术和real-time PCR等方法检测了过表达Gpr3对猪卵泡颗粒细胞增殖及凋亡的影响. 结果显示,过表达Gpr3后,猪颗粒细胞的增殖水平显著下调,G0/G1期细胞的百分比增加,S期细胞减少,Cyclin B1和CDK1 mRNA的表达量也显著降低;同时,显著增加了颗粒细胞的凋亡率,在抑制Bcl-2表达的同时,促进了Bax的表达. 结果表明：过表达Gpr3在猪颗粒细胞中具有抑增殖促凋亡的作用,丰富了其在调节卵泡发育过程中的生物学功能.     

重组人卵透明带ZP3蛋白在毕赤酵母中的表达

利用P.pastoris表达人卵透明带ZP3蛋白。设计特定引物从全长hZP3 cDNA上扩增含跨膜区序列的人卵透明带ZP3基因片段,并在N末端接上串联组氨酸编码序列的重组基因序列;扩增片段插入表达载体pPIC9K中;线性化后的重组质粒转入P.pastoris中,用高浓度G418筛选高拷贝菌株,然后甲醇诱导目的蛋白表达。用SDS-PAGE和Western blot分析表达产物。结果发现P.pastoris表达的人ZP3蛋白可以分泌到培养液中,并且可溶性好。纯化前后的重组人ZP3蛋白均能与兔抗猪ZP3蛋白抗体发生交叉反应,证实表达的目的蛋白具有反应原性。     

Expression of mRNA for activin-binding protein (follistatin) during early embryonic development of Xenopus laevis.

Follistatin is a specific activin-binding protein and is supposed to control activin functions. During Xenopus embryonic development, activin is thought to act as a natural mesoderm-inducing factor. We isolated here the Xenopus follistatin cDNA from Xenopus ovary cDNA library and studied the expression of Xenopus follistatin gene during the course of early embryonic development. The Xenopus follistatin has an 84% homology at the level of deduced amino acid sequence with human and porcine follistatin. Its 3.5 kb mRNA is first expressed at the gastrula stage, when the expression of activin mRNA becomes first detectable, and increased thereafter. Another species of 2 kb mRNA become detectable from early neurula and also increased dramatically in tadpole. These results suggest that the follistatin acts also as a regulator of activin in inductive interactions during amphibian embryonic development.     

Follistatin gene expression in the ovary and extragonadal tissues

Follistatin is a glycosylated single-chain protein originally isolated from porcine follicular fluid. It specifically inhibits the secretion of FSH from the pituitary. We have now isolated and characterized a cDNA for rat follistatin from the PMSG-stimulated ovarian library. The deduced amino acid sequence of the rat follistatin precursor is highly homologous (greater than 98%) to porcine and human follistatins including potential Asn-glycosylation sites. The genomic clone encoding rat follistatin was also isolated and revealed that the exon and intron organization of the follistatin gene structure is conserved among rat, porcine, and human. Northern analyses in rat tissues demonstrated that the follistatin gene is expressed not only in the ovary but also in the kidney and brain. In the immature rat ovary, the follistatin mRNA level is stimulated by PMSG injection (20 IU/rat), but is not affected by human CG (10 IU/rat) after PMSG administration. In situ hybridization studies revealed that the mRNA level in the ovary was low in primordial follicles, but dramatically increased in the granulosa cells of the growing secondary and tertiary follicles and then decreased in the mature preovulatory follicles. A strong follistatin mRNA signal was observed over the collecting tubules of the outer medulla of the kidney, and a weak to moderate signal was detected in brain. The broad tissue distribution of follistatin mRNA strongly suggests other physiological roles for follistatin besides the inhibition of pituitary FSH release.     

绵羊Follistatin基因表达及其结构域的功能分析

为研究羊Follistatin基因的功能,提取了绵羊卵巢总RNA,通过RT-PCR方法获得羊Follistatin cDNA的完整开放阅读框 (1 038 bp)。去除信号肽序列后与原核表达载体pET41a连接,构建重组表达质粒pFSsig?,经大肠杆菌诱导表达获得FS sig?蛋白 (66 kDa)。通过RT-PCR克隆了包含N端和结构域1的Follistatin突变体 (FS N+D1),将FS N+D1片段插入慢病毒载体 (pLEX-MCS) 构建了pFS-N+D1慢病毒重组表达质粒。在293T细胞中进行慢病毒的包装,再感染绵羊肌肉原代细胞,得到稳定表达FS N+D1的肌肉细胞系,通过细胞生长曲线结果显示稳定表达FS N+D1的肌肉细胞明显比正常肌肉细胞增殖快,且差异极显著 (P＜0.01),表明绵羊FS N+D1结构域有促进肌肉细胞生长的功能。     

可溶性HLA-A*0203-BSP原核表达载体的构建及其在大肠杆菌中的高效表达

目的：构建HLA-A*0203重链胞外域羧基端融合生物素化酶BirA底物肽(BSP)的融合蛋白（HLA-A*0203-BSP）的原核表达载体并在大肠杆菌中进行表达。方法：以RT-PCR方法从HLA-A2+ 供者外周血单个核细胞(PBMC)中克隆HLA-A*0203重链基因的cDNA并测序鉴定,然后以PCR方法构建HLA-A*0203-BSP的原核表达载体,在大肠杆菌BL21(DE3)菌株中诱导表达并以免疫印迹鉴定。结果：DNA测序显示,从3名HLA-A2+ 供者PBMC中克隆的cDNA中,只有从供者2获得编码HLA-A*0203重链基因的cDNA。将编码重链胞外域1-276的序列和编码BSP的序列融合,构建HLA-A*0203-BSP融合蛋白的原核表达载体并经测序验证。该融合蛋白在BL21(ED3)中获得高效表达,约占菌体总蛋白的30％;产物相对分子质量约为34 kD,与理论大小一致。Western印迹分析显示融合蛋白完全存在于包涵体中。结论：成功克隆HLA-A*0203重链基因的cDNA,构建HLA-A*0203-BSP融合蛋白的原核表达载体,并在大肠杆菌中获得高效表达,为制备HLA-A*0203四聚体打下基础。     

猪A组轮状病毒Vp6基因与乳酸菌非抗性表达载体的重组及在大肠杆菌中的表达

以猪A组轮状病毒mRNA为模板,应用反转录聚合酶链式反应(RT-PCR)技术,扩增了1194bp的Vp6基因,通过T-A克隆技术,将PCR产物克隆至克隆载体pGEM-TVector中,构建克隆质粒pGEM-T-Vp6。用SacⅠ和KpnⅠ双酶切pGEM-T-Vp6和以胸苷酸合成酶基因(thymidylate synthase,thyA)为选择压力的非抗生素抗性的穿梭表达载体pW425t,并将纯化的Vp6基因亚克隆至表达载体pW425t中,构建出可以在乳酸菌与大肠杆菌之间穿梭表达的原核表达重组质粒pW425t-Vp6。将pW425t-Vp6转化至thyA基因缺陷型的大肠杆菌感受态E.coliX13中,经生长功能弥补筛选阳性克隆,通过SDS-PAGE分析,可见约44.88kD的融合蛋白。由Western blot分析,表明该蛋白具有与轮状病毒多克隆抗体的反应原性,从而为pW425t-Vp6在乳酸菌受体菌株中表达提供理论基础和实验依据。     

Cloning,sequencing, and expressional analysis of the chick homologue of follistatin

Follistatin, a secreted glycoprotein, has been shown to act as a potent neural inducer during early amphibian development. The function of this protein during embryogenesis in higher vertebrates is unclear, and to further our understanding of its role we have cloned, sequenced, and performed an in-depth expressional analysis of the chick homologue of follistatin. In addition we also describe the expression pattern of activin βA and activin β B, proteins that have previously been shown to be able to interact with follistatin. In this study we show that the expression of follistatin and the activins do not always overlap. Follistatin was first detected in Hensen's node and subsequently in the region described by Spratt [1952] as the neuralising area. In older embryos it was also expressed in a highly dynamic manner in the hind-brain as well as in the somites. We also present evidence that follistatin may have a later role in the resegmentation of the somites. We were unable to detect the expression of activin βA during early embryogenesis, whereas activin βB was first expressed in the extending primitive streak and subsequently in the neural folds. The results from this study are consistent with a role for follistatin in neural induction but suggest it has additional functions unrelated to its inhibitory actions on activins. © 1995 Wiley-Liss, Inc.     

Follistatin inhibits activin-induced differentiation of rat follicular granulosa cells in vitro.

The effect of follistatin on activin-induced granulosa cell differentiation was investigated in freshly harvested granulosa cells from diethylstilbestrol (DES)-treated rats. Activin induced a remarkable change in granulosa cellular morphology from elongated fibroblast-like to round cells, which follistatin prevented. Follistatin itself had no influence on the cellular morphology. We studied the action of follistatin on activin-induced differentiation of granulosa cells cultured in a chemically defined medium. Addition of activin (30 ng/ml) to the culture increased the FSH binding site approximately 2-fold compared with the control (spontaneous expression) level, whereas follistatin reduced the activin-induced expression level to the control level in a concentration-dependent manner. Activin (30 ng/ml) markedly augmented FSH-induced hCG binding and progesterone production by approximately 20-fold, and these effects were suppressed by follistatin in a concentration-dependent manner. Similarly, addition of follistatin to the culture induced a concentration-dependent decrease of activin-enhanced inhibin-producing activity, but had no effect on FSH-induced inhibin production. These results suggest that follistatin/activin-binding protein binds to activin stoichiometrically to suppress the activin-induced differentiation of rat granulosa cell in vitro, but follistatin itself has no direct effect on activin-independent reactions.