新加坡石斑鱼虹彩病毒ORF39L基因克隆、表达及抗体的制备

为研究新加坡石斑鱼虹彩病毒(Singapore grouper iridovirus,SGIV)核心基因ORF39L在病毒侵染过程中的作用,制备了SGIV ORF39L表达蛋白的抗体;通过生物信息学软件预测SGIV ORF39L的抗原表位基因,并以所选抗原表位基因片段39Ag1和39Ag2构建原核表达质粒pET32a-39Ag1和pET32a-39Ag2;将质粒转入大肠杆菌BL21(DE3)中成功表达融合蛋白pET32a-VP39Ag1和pET32a-VP39Ag2;用所得融合蛋白分别免疫小鼠,获得了高效特异抗体39Ab1和39Ab2;利用制备的抗体进行间接免疫荧光试验,结果显示,VP39参与了SGIV的装配。本研究结果为进一步研究ORF39L基因在SGIV感染过程中的作用机制提供了数据资料。     

刺参C型凝集素(AJL)基因的克隆及原核表达分析

为了开发海参免疫基因,生产重组免疫因子蛋白,利用cDNA末端快速扩增技术(Rapid-amplification of cDNA ends,RACE)获得了刺参凝集素基因(Apostichopus japonicus Lectin,AJL)的编码区序列(Coding sequence,CDS),CDS的长度为492 bp,编码163个氨基酸,其中成熟肽由143个氨基酸组成。将成熟肽的基因克隆并连接至载体p ET-32a(+)中,成功构建了原核表达的重组体p ET-32a(+)-AJL,进一步将重组体转化至大肠杆菌感受态细胞BL21(DE3)中,经诱导表达、分离纯化,获得了融合表达蛋白。本研究结果为进一步研究重组刺参C型凝集素在刺参健康养殖上的应用奠定了基础。     

新城疫病毒7793株HN基因片段原核表达及功能鉴定

HN蛋白是新城疫病毒(Newcastle disease virus,NDV)的血凝素神经氨酸酶蛋白(hemagglutinin-neuraminidase,HN),是NDV识别宿主细胞的包膜蛋白。NDV-HN能与NK细胞的NKp46受体结合,活化NK细胞上调TNF-α和IFN-γ发挥抗瘤效应[1]。然而HN蛋白是否能直接活化NK细胞上调TNF相关凋亡诱导配体(TNF-related apoptosis-inducing ligand,TRAIL)尚待阐明。因此对新城疫病毒7793株(NDV 7793)HN基因主要抗原表位区域进行克隆和原核表达,并对重组HN(recombinant HN,rHN)蛋白是否能活化NK细胞上调TRAIL进行研究。流式细胞分析表明rHN蛋白的纯化产物能与小鼠胸腺的NK细胞膜分子NKp46结合,并上调NK细胞TRAIL的表达。HN抗体阻断试验能部分抑制HN-NKp46结合及NK细胞TRAIL上调的效应。这一结果将为进一步研究NDV-HN直接活化NK细胞增强抗肿瘤效应的胞内信号传导提供依据。     

太平洋鳕抗冻基因AFP4的原核表达及多克隆抗体的制备

为进一步研究太平洋鳕Gadus macrocephalus抗冻蛋白AFP4的功能,通过RT-PCR方法克隆得到太平洋鳕4型抗冻蛋白基因(AFP4)的开放阅读框(open reading frame,ORF),构建原核表达载体并优化表达条件,利用纯化的重组蛋白制备多克隆抗体,采用间接ELISA法检测抗体的效价,用Western-blot法分析重组蛋白的免疫原性。结果表明:AFP4基因编码区长度为375 bp,编码125个氨基酸;将AFP4基因的ORF与载体p ET-32a连接,构建重组体p ET-32a-AFP4,并将其转入大肠杆菌BL21(DE3)感受态细胞中;经诱导、表达和条件优化,发现该重组体在37℃、0.01 mmol/L IPTG条件下诱导3 h获得最大的表达量;对该重组蛋白进行纯化,利用纯化的蛋白免疫新西兰大白兔制备多克隆抗体,采用间接ELISA法检测该抗体效价为1∶1 600 000;Western blot分析显示,重组蛋白可以与兔抗AFP4多克隆抗体发生特异性结合,表明该重组蛋白具有免疫原性。本研究结果可为深入研究太平洋鳕AFP4蛋白功能提供理论依据。     

藏绵羊乳腺溶菌酶基因的克隆、原核表达及抗菌活性研究

克隆了藏绵羊溶菌酶(LZM,EC 3.2.1.17)基因cDNA序列,构建重组质粒pET-32a-LZM,并在大肠杆菌BL21中成功表达,重组LZM有一定的抗金黄色葡萄球菌活性。系统进化树分析表明,藏绵羊乳腺LZM与山羊LZM的亲缘关系最近。实时荧光定量PCR(RT-qPCR)结果表明,藏绵羊乳腺LZM在乳腺中表达量最大。此结果对LZM的应用有一定价值。     

奥利亚罗非鱼促性腺激素释放激素及相关肽基因的克隆与表达

采用RT-PCR方法从奥利亚罗非鱼丘脑中扩增出长约386 bp的目的序列GnRH/GAP,先将其克隆到T载体上,通过酶切鉴定、序列测定分析,确认序列的正确性后,将此片段克隆到表达载体pMAL-c2x中构建重组表达质粒pMAL-GnRH/GAP,再转化到大肠杆菌TB1中,经IPTG诱导后成功表达出与预期大小相符的相对分子量约56 000的融合蛋白。     

丹波黑大豆醛脱氢酶基因GmALDH3-1的克隆、原核表达和功能分析

醛是一类具有高度反应活性的毒性物质,在生物体中主要由膜脂过氧化,氨基酸氧化和蛋白质的糖基化产生。醛脱氢酶(aldehyde dehydrogenase,ALDH)是一类以多种醛类为底物的酶类,醛脱氢酶基因是一类编码将醛脱氢氧化为相应羧基酸的基因,在植物,动物和微生物中均有发现。通过RT-PCR方法从丹波黑大豆根中扩增出基因GmALDH3-1。构建原核表达载体pGEX-4T-1-ALDH3-1,在E.coli BL21中表达,并研究不同浓度IPTG、时间和温度对ALDH3-1蛋白表达的影响,结果表明28℃下诱导6h ALDH蛋白表达量最大,而IPTG浓度对ALDH3-1的表达量影响不大。在添加有不同浓度Al、Cu和Cd的液体LB培养基中培养转化菌和对照菌,检测转化菌和对照菌的生长曲线,生长曲线实验结果表明ALDH3-1转化工程菌对上述金属离子具有一定的耐受性。这些结果为进一步研究其结构和生物学功能奠定了基础。     

太平洋鳕神经坏死病毒衣壳蛋白(CP)的原核表达及条件优化

为深入研究太平洋鳕Gadus macrocephalus神经坏死病毒(Pacific cod nervous necrosis virus,PCNNV)衣壳蛋白(capsid protein,CP)的功能,将PCNNV cp基因连接到p ET-32a原核表达载体中,构建原核表达重组载体p EVCP,将其转化至大肠杆菌E.coli BL21(DE3)表达系统中进行融合表达,对诱导剂IPTG浓度、诱导时间和温度等诱导表达条件进行优化,采用SDS-PAGE及质谱技术鉴定表达产物。结果表明:本研究中成功构建了重组载体p EVCP,表达的目的蛋白相对分子质量约为55 000;目的蛋白经质谱鉴定,有6个肽段的氨基酸序列与预期一致;对重组菌株p EVCP/BL21的最佳诱导条件为IPTG浓度0.1 mmol/L、最佳诱导时间3 h、温度30℃。本研究结果可为后续冷水鱼类病毒疫苗的研制及病毒快速检测试剂盒的研发奠定基础。     

GST-CTCF融合蛋白原核表达载体的构建及表达

目的构建人CCCTC结合因子(CCCTC-binding factor,CTCF)与谷胱甘肽转移酶(glutathione S-transferase,GST)重组蛋白的原核表达载体,并进行诱导表达。方法以pEASY-T1-SIMPLE-CTCF为模板,设计引入限制性酶切位点的CTCF引物,PCR方法扩增目的片段,产物经限制性内切酶酶切后与原核表达载体pGEX-4T-1连接,构建成pGEX-4T-1-CTCF原核表达载体,转化至大肠埃希菌BL21中,经异丙基硫代-β-D半乳糖苷(IPTG)进行诱导,Western blot检测GST-CTCF融合蛋白的表达情况。结果经菌液PCR、质粒双酶切分析、基因测序分析证实重组表达质粒pGEX-4T-1-CTCF构建成功,GST-CTCF融合蛋白产物经Western blot鉴定为特异性表达。结论成功构建了pGEX-4T-1-CTCF原核表达质粒,获得特异性的GST-CTCF融合蛋白,为进一步研究转录因子CTCF的功能奠定了基础。     

微生物源纤溶酶基因的克隆及表达研究进展

血栓栓塞性疾病严重危害人类生命和健康,溶栓疗法效果显著。在新型溶栓制剂的研究中,微生物来源的纤溶酶研究引起越来越多的关注。通过分子生物学手段构建重组纤溶酶,实现酶的高效表达已经成为研究热点,本文对微生物来源的纤溶酶基因克隆与表达的研究现状进行了综述。     

中国劳动力迁移的综合理论框架研究

劳动力区际迁移是经济学、区域科学以及经济地理学的重要研究领域。学术界提出了许多理论来解释劳动力为什么从一个区域迁移到另一区域。本文在介绍和批评新古典劳动力迁移模型、人力资本理论以及行为迁移理论的基础上,结合有关实证研究,提出一个解释中国劳动力区际迁移的理论分析框架。     

Changes in dietary bioaccumulation of tributyltin chloride (TBTCl) in red sea bream (Pagrus major) with the concentration in feed

The effect of the concentration of tributyltin (TBT) in feed on the dietary bioaccumulation of tributyltin chloride (TBTCl) was studied in an 8-week uptake experiment and a 4-week elimination experiment using red sea bream (Pagrus major). The biomagnification factor (BMF) and the assimilation efficiency (AE) decreased from 0.30 to 0.15 and from 13% to 5.9%, respectively, as the TBT concentration in feed increased from 1.3 to 20 microg/g. The elimination rate constant (k(2)) was independent of the TBT concentration in the fish. Laboratory measurements of the BMF and AE of TBTCl underestimate actual field values if highly contaminated feed is used. Judging from the BMF and AE, the risk of the bioaccumulation of TBTCl through the food chain might be smaller than that of polychlorinated biphenyls.     

牦牛DIO2基因克隆及其在骨骼肌的表达分析

为探索DIO2基因调控牦牛骨骼肌发育及肌纤维类型组成的潜在功能,克隆DIO2基因CDS序列并进行相关的生物信息学分析,以及比较分析其在牦牛骨骼肌的差异表达.结果显示,DIO2基因的CDS全长为789 bp,编码132个氨基酸.序列同源性比对及系统进化树分析发现,牦牛DIO2基因与普通牛的亲缘关系最近,核酸及蛋白序列与普通牛一致;与小鼠的亲缘关系较远,序列同源性仅为86.75%和87.12%,与原鸡的亲缘关系最远,二者的同源性分别为79.17%和76.30%.理化性质预测表明DIO2蛋白为不稳定疏水性蛋白,有跨膜结构域,具有24个磷酸化位点,蛋白结构为无规则卷曲、α-螺旋和延伸链三者交替出现.采用实时荧光定量PCR分析显示,DIO2基因在牦牛的脾、肺等内脏组织表达无显著差异;在背最长肌和半腱肌的表达量显著高于脂肪和肝脏组织(P<0.01).此外,DIO2在金川牦牛半腱肌的表达量显著高于麦洼牦牛(P<0.05).以上结果为研究DIO2在牦牛骨骼肌的功能提供参考资料.     

牦牛Caspase-3克隆、序列分析与组织表达研究

半胱天冬酶3(Aspartate-specific cysteinyl proteinase 3,Caspase-3)是哺乳动物最重要的细胞凋亡执行者,为探究Caspase-3基因在牦牛繁殖活动中的调控作用奠定基础,试验对牦牛Caspase-3编码区进行克隆和生物信息学分析,并比较牦牛和黄牛下丘脑、垂体、卵巢、输卵管及子宫等组织的表达差异.结果表明:牦牛Caspase-3基因编码区全长834 bp,编码277个氨基酸,与黄牛Caspase-3基因相比同源性最高(99. 52%)且遗传距离最近,发生4个碱基突变;与鸡同源性最低(71. 32%)且遗传距离最远,保守且符合动物进化进程.q PCR结果显示Caspase-3在牦牛生殖轴中的表达量均高于黄牛,其中,在下丘脑、输卵管中达到P <0. 01水平,在子宫中达到P <0. 05水平.推测极端环境应激引起Caspase-3基因的高表达可能影响母牦牛的繁殖性能.     

山羊ZP3基因的cDNA克隆、序列分析及组织表达研究

对高繁金堂黑山羊和低繁藏山羊ZP3基因c DNA进行克隆、序列分析,并采用Real-time PCR技术对其在发情前期母羊卵巢组织的表达进行研究.结果表明:山羊ZP3基因编码区全长为1269bp,共编码422个氨基酸,两品种间有2处碱基差异,但未导致氨基酸的差异.山羊的ZP3基因在卵巢表达量高,与其他组织差异极显著(P<0.01),但两个品种间差异不显著(P>0.05).说明ZP3基因主要在卵巢中表达,但可能不是影响山羊多羔性状的主基因.     

Biomagnification of organochlorine pollutants in farmed and wild gilthead sea bream (Sparus aurata) and stable isotope characterization of the trophic chains

Organochlorine pollutants (pesticides and polychlorinated biphenyls) were analysed in farmed and wild gilthead sea bream (Sparus aurata) tissues (white muscle and liver) from the Western Mediterranean (Spain) and in their diets. Determination was carried out by gas chromatography coupled to tandem mass spectrometry after clean up of the fatty extracts by normal phase HPLC, with detection limits around 0.1 ng/g. Carbon and nitrogen stable isotope ratios were also determined in the samples. Organochlorine compounds concentration was found to be uniform throughout the year in farmed fish, in both white muscle and liver. In contrast, wild fish showed contamination profiles that reflect environmental factors and the biological cycle. Although biomagnification factors for white muscle and liver were found to be 2.4 and 3.0, respectively for farmed fish, and 0.15 and 0.54 for wild specimens, wild fish presented higher levels of organochlorine contaminants than farmed fish. Nitrogen stable isotopes determination in muscle from wild and farmed sea bream during the year gave us a profile related to the biological cycle. delta(15)N mean values from farmed fish were 2.0 per thousand higher than from wild fish throughout the year that corresponding to close to one trophic step. delta(13)C values were stable during the year, and also more enriched in the case of farmed fish. The low levels of contaminants found in the feed supplied to farmed fish explain the organochlorine concentrations in their tissues which remain below wild fish, in spite of the intensive culture conditions and higher trophic level of cultured specimens.     

The influence of the In(Lu) gene on expression of CDw75 antigens on human red blood cells

The influence of the In(Lu) gene on human red blood cell (RBC) expression of CDw75 antigens was examined. CDw75 antigens were increased in expression on Lu(a-b-) cells of the dominant inhibitor type in comparison with red cells from donors of other Lutheran (Lu) phenotypes. In contrast, CD44 epitopes detected with F10-44-2, A3D8 and BU52 monoclonal antibodies (mAb) were decreased in expression on Lu(a-b-) red cells. Among normal blood donors of the common phenotype Lu(a-b+) there was a wide range of expression of CDw75 antigens on red cells. The results show that CDw75 is a quantitative polymorphism of human red cells and, among antigens influenced by the In(Lu) gene, is unique in being up-regulated in expression.     

Trace elements in the muscle of red shrimp Aristeus antennatus (Risso, 1816) (Crustacea, Decapoda) from Ligurian sea (NW Mediterranean): variations related to the reproductive cycle

Concentrations of total and organic mercury, cadmium, lead, copper, iron, manganese and zinc were determined in the muscular tissue of 135 specimens of Aristeus antennatus (Risso, 1816) collected during a 3-year period in the Ligurian sea (NW Mediterranean). The aim of the present work was to study the relationships between the concentrations of these trace elements and the main biological parameters characterising the organisms, i.e. sex and size with special attention to the sexual maturity of the female specimens. Mercury is the only element showing a significant correlation with the size of the specimens, and for this reason it could give some indications about the age of the shrimps. However, comparison between females collected at four different reproductive phases shows that reproductive individuals are characterised by higher levels of mercury with respect to non-reproductive individuals of the same size.