猪肠病毒G型部分VP1蛋白的原核表达及多克隆抗体制备

本研究扩增猪肠病毒G型(EV-G)的部分VP1基因,将其克隆至原核表达载体,并对其进行诱导表达,获得重组蛋白并制备多克隆抗体,通过酶联免疫吸附试验,免疫印迹试验和间接免疫荧光试验对其进行验证。结果表明,本研究制备的兔抗EV-G-VP1多克隆抗体具有良好的免疫原性和反应性,可以特异性识别EV-G感染Marc-145细胞表达的VP1蛋白,为猪肠病毒G型免疫学检测方法的建立提供良好的生物学材料。

基于EXPRESS-G的BAS信息模型拓扑结构浅析

基于智能建筑中典型的BAS,在CAD设计的基础上,将EXPRESS-G图形语言运用在BAS设计过程中,以达到在设计与施工过程中减少重复劳动,简化设计过程,降低成本的目的。建立一个包含设备列表、网络拓扑结构、以及点表和操作序列的BAS信息模型,基于此模型可以在建筑的全生命周期中使BAS模型在符合IFC标准的前提下保持单向传递性,利用创建出的模型可以直接使用能耗仿真及维修管理系统等的应用程序,并且能在调试和使用过程中直接从中提取各自所需的数据信息。

EXPRESSION OF cDNA FOR RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR IN ESCHERICHIA COLI AND CHARACTERIZATION OF THE PROTEIN

Objective: To determine the biological activity of rhG CSF and it's characterization Methods: The prokaryotic expression vector pG01 containing human G CSF cDNA were constructed with DNA recombination technology Results: We had achieved high level expression of the human G CSF in E coli , where it represented at least 23 6% of the total protein as determined from SDS PAGE gels The human G CSF was expressed as inclusion bodies in E coli The inclusion bodies were solubilized in a solution containing 7M urea, renatured by dialysis, isolated and purified by DEAE sepharose CL 6B ion exchange and Superdex 75 gel filtration chromatography The purified rhG CSF was confirmed by coincidence of biological activity and protein demonstrated by SDS PAGE It was homogeneous with respect to mol Wt (18400) The purity of the rhG CSF might be >90 per cent Conclusion: The purified rhG CSF in our laboratory had dramatically the biological activity of regulating proliferation and differentiation of the human G CSF dependent cell line NSF 1 and the progenitor cells of granulocytes of human bone marrow

Prokaryotic Expression of Glycoprotein Gene of Infectious Hnematopoietic Necrosis Virus and Polyclonal Antibody Preparation

[Objective]The aim is to perform prokaryotic expression of the glycoprotein gene of infectious hnematopoietic necrosis virus and polyclonal antibody preparation. [Methods]Glycoprotein gene( G) of infectious hematopoietic tissue( IHNV) was synthesized,cloned to prokaryotic expression system pET-30a vector,yielding the recombinant plasmid pET-30a-IHNV-G. The yielded pET-30a-IHNV-G was transformed into E. coli strain BL21( DE3) plySs. [Results] SDSPAGE and Western blot results showed that protein G successfully expressed in E. coli at 37 ℃,1 mmol /L IPTG induction for 4 h. The molecular weight of fusion G protein was 57 KD. The polyclonal antibody was prepared by immunizing mice with the product of gel purification. ELISA analysis showed that the serum titer reached 1∶10 000. [Conclusion]The expressed G protein and the serum with polyclonal antibody obtained in this study provided the theoretical basis for the development of IHNV vaccine and detection of colloidal gold test strip.

大口黑鲈弹状病毒G蛋白胞外区原核表达及多克隆抗体制备

【目的】获得能特异性识别大口黑鲈弹状病毒(Micropterus salmoides rhabdovirus, MSRV)G蛋白的多克隆抗体,为后续MSRV双抗夹心法胶体金试纸条的研制提供材料。【方法】根据GenBank上MSRV G蛋白的基因序列(GenBank登录号:OK272491.1)设计扩增其胞外区的特异性引物,以感染MSRV的GS细胞的cDNA为模板,通过PCR扩增获得目的序列,双酶切后连接至pET-28a(+)表达载体构建重组质粒pET-28a-ΔG,将重组质粒转化大肠杆菌Trans5α感受态细胞。挑取测序正确的阳性克隆提取重组表达质粒pET-28a-ΔG,转化大肠杆菌BL21(DE3)感受态细胞,再次挑取阳性克隆,测序正确后用IPTG诱导表达。目的蛋白经Ni-NTA树脂层析柱纯化后免疫BALB/c雌性小鼠,4次免疫后眼眶采血获得抗MSRV G蛋白的多克隆抗体,最后通过ELISA、Western blotting和间接免疫荧光试验(IFA)鉴定多克隆抗体的效价和特异性。【结果】PCR结果显示,获得大小为1 323 bp的目的条带。SDS-PAGE结果显示,构建的重组表达质粒可高效表达目的蛋白,Ni柱纯化后获得单一目的蛋白,分子质量在48.5 ku左右,与预期相符,且纯度符合免疫原要求。ELISA结果显示,制备的多克隆抗体效价为1∶204 800。Western blotting结果显示,制备的多克隆抗体可特异性识别不同批次的MSRV G蛋白。IFA结果显示,制备的多克隆抗体能特异性识别天然状态下的G蛋白。【结论】本研究利用MSRV G蛋白的胞外区重组蛋白成功制备出能特异性识别MSRV G蛋白的多克隆抗体,为后续MSRV的免疫检测和疫苗开发奠定了基础。

Gene Cloning and Expression Analysis of G Protein αq Subunit from Helicoverpa assulta (Guenée)

The cDNA encoding the G protein αq subunit was isolated from the antennae of Helicoverpa assulta （Guen6e） by reverse transcription polymerase chain reaction （RT-PCR） and named as HassGαq. Sequencing analysis showed that the fulllength of HassGαq open reading frame （ORF） is 1 062 bp, 353 amino acid residues are encoded. The predicted molecular weights （MW） and isoelectric point （PI） are 41.5 kD and 5.15, respectively. HassGαq gene was then constructed into expression vector pGEX-4T-2 for over expression in prokaryotic cells. The SDS-PAGE and Western blot analysis showed that induced by Isopropyl-β-D-Thiogalactoside （IPTG）, the GST-HassGαq fusion protein is expressed in Escherichia coil BL21, and its MW was found to be about 66 kD nearly equal to the predicted. In addition, RT-PCR analysis showed that the expressions of HassGαq are not tissue specific.

EFFECT OF ELECTROACUPUNCTURE ON GENE EXPRESSION OF α- SUBUNIT OF Go-PROTEIN IN THE HIPPOCAMPUS OF RATS WITH HYPERTENSIVE CEREBRAL HEMORRAGE

Objective: To observe the effect of electroacupuncture (EA) on gene expr ession o f α subunit of Go-protein in the brain of rats with hypertensive cerebral hem or rage and study its underlying mechanisms of EA in ameliorating cerebral hemorrag e. Methods: A total of 130 SD rats were randomly divided into nor mal control gro up (n=10), sham operation group (n=40), model group (n=40) and EA group (n=40). The latter 3 groups were further divided into 6 h, 24 h, 48 h and 72 h (tim e course s) subgroups, with 10 rats being in each subgroup. The hypertensive cerebral hem orrage model was induced by injecting 1 μL of collagenase (0.5 U/μL collagena se Type Ⅶ) and heparin (7 U/μL) into the caudate nucleus in rats with renovascul ar hypertension (by clipping the bilateral renal arteries). The gene expression of α subunit of Go-protein in the hippocampus tissue of rats was detected with No rthern blotting hybridization analysis. EA (continuous waves, 120 pulses/min in frequency, 1 mA in intensity and duration of 30 min) was applied to "Shuigou" (水沟 GV 26), bilateral "Neiguan"(内关 PC 6) and bilateral "Housanli"(Zusanl i, 足三里 ST 36). Results: The gene expression of α subunit of Go-protein in th e hippocampus tis sue of the rats was obviously downregulated in hypertensive cerebral hemorrage m odel group and significantly upregulated after EA treatment wit h the extension of time. Conlusion: EA may relieve cerebral hemorr age by regulating the gene transcription of α subunit of Go-protein and incre asing the expression of Go-α protein. This may be one of the molecular mechani sm s of EA in improving hypertensive cerebral hemorrhage.

Gene Cloning and Tissue-Specific Expression of G Protein β Subunit in Microplitis mediator (Hymenoptera: Braconidae)

A gene encoding a novel G protein β subunit of β1 subclass, GβMmed was isolated from Microplitis mediator （Hymenoptera： Braconidae）. The full-length sequence of GβMmed is 1 119 bp, the cDNA contains a 1 023 bp open reading frame that encodes a protein with 340 amino acids, and the predicted molecular weight of GβMmed is 37.23 kDa and isoelectric point is 5.86. By the quantitative real-time RT-PCR method, the tissue-specific expression and quantitative changes in the developmental expression profile of GβMmed were detected. It was found that GβMmed was abundantly expressed in M. mediator antennae, head （without antennae）, thorax, abdomen, legs and the wings, and especially at high levels in abdomen. In antennae, expression varied through 1st day before emergence to 5-d-old adults, and had equal expression levels detected in females and males in total. In head, GβMmed expresses while initially high in females, and have another peaked in stage 4 and 1st day, in males showed a peak of GβMmed expression prior to emergence and relatively low levels after emergence. In female abdomen GβMmed expression levels have two peaks in stage 1 and the 5th d, but just have one peak in male abdomen in stage 1. In all other tissues expression was low and stable.

重组链球菌蛋白G的SUMO融合表达策略和活性表征

目的:链球菌蛋白G(Streptococcus Protein G,SPG)是一种能够和多种哺乳动物的IgG相结合的蛋白质,广泛用于重组蛋白和抗体的纯化,目前市场产品主要为His-SPG,本研究拟采用类泛素蛋白修饰分子(Small Ubiquitin-Like Modifier,SUMO)融合表达SPG,以提高SPG产量,降低纯化柱料的生产成本。方法:将SPG基因分别连接至pET28a和pSmartⅠ质粒,经大肠杆菌重组表达两种蛋白,并比较产量、纯度和活性的差异。结果:His-SUMO-SPG产量为12.5 mg/L,较His-SPG(8.4 mg/L)提高约49%;包被两种SPG于96微孔板作为固定相,检测其捕获兔多抗和鼠单抗能力,His-SUMO-SPG的检测信号(OD_(450))较His-SPG更高,具有更高载量;生物素化His-SPG对小鼠的IgG1、IgG2a、Ig2b以及兔IgG最低检测浓度分别为0.703 pmol/L、0.098 pmol/L、0.524 pmol/L和0.927 pmol/L,生物素化His-SUMO-SPG最低检测浓度分别为0.887 pmol/L、0.084 pmol/L、0.667pmol/L和0.865 pmol/L,两种SPG最低检出限接近。结论:His-SUMO-SPG和His-SPG在检测性能方面比较接近,His-SUMO-SPG结合IgG载量略高于His-SPG,且His-SUMO-SPG产量更高,SUMO融合表达蛋白G方法为工业大规模生产提供了一种有效策略。

电压门控钾通道亚家族G成员1在肺癌中的表达及生物学作用

目的:探讨电压门控钾通道亚家族G成员1(potassium voltage-gated channel subfamily G member 1,KCNG1)在人肺癌组织中的表达水平和临床意义以及其在肺癌细胞恶性生物学行为中的作用。方法:通过TCGA数据库(The Cancer Genome Atlas)分析KCNG1 mRNA在人肺癌组织中的表达水平,探究其与肺癌患者临床病理特征及预后的关系;基于KCNG1 mRNA表达水平构建预测肺癌患者预后的列线图模型;采用基因本体(GO)功能富集分析和京都基因与基因组百科全书通路(KEGG)富集分析探究KCNG1潜在的生物学功能;采用基因集富集分析(GSEA)预测KCNG1相关差异表达基因参与调控的相关信号通路。采用实时荧光定量PCR(qRT-PCR)及蛋白免疫印迹分别检测肺癌A549和H1299细胞系以及正常肺上皮细胞中KCNG1 mRNA和蛋白表达水平;通过转染siRNA构建KCNG1敲减的细胞株,分别采用EdU,Transwell,Matrigel Transwell,细胞划痕愈合及血管生成拟态实验检测细胞株生物学行为变化。结果:KCNG1 mRNA在肺癌组织中显著高表达且与肺癌患者不良预后密切相关(P均<0.05);列线图模型初步证实KCNG1可能是肺癌潜在的生物标志物,并具有良好的预后评价功能;GO及KEGG富集分析提示KCNG1在调节激素分泌、离子转运、神经肽信号通路及细胞间黏附等生物学过程中发挥重要作用;GSEA结果提示KCNG1相关差异表达基因主要富集在KRAS,MTORC1,MYC,P53及WNT信号通路;KCNG1敲低的肺癌细胞增殖、迁移、侵袭和成管能力明显受到抑制(P均<0.05)。结论:KCNG1 mRNA在肺癌中显著高表达且与患者不良预后密切相关;siRNA介导的KCNG1敲低可显著抑制肺癌细胞的恶性生长。

扎那纹胸鮡g型溶菌酶的cDNA克隆及组织表达分析

目的鉴定扎那纹胸鮡g型溶菌酶(GzLysG)编码序列,分析其在宿主免疫系统中的作用。方法通过巢氏PCR技术克隆了GzLysG cDNA序列,通过ExPASy、SignalP 5.0、CDD、Clustal Omega等在线软件对GzLysG蛋白进行生物信息学分析,利用RT-qPCR技术分析了GzLysG mRNA在不同组织中的表达情况。结果GzLysG cDNA的开放阅读框全长558 bp,编码185个氨基酸,其蛋白序列分子量为20478.20 u,理论等电点为9.16,是一种稳定的碱性亲水性蛋白质。该蛋白无信号肽,含有g型溶菌酶典型的催化活性位点、GEWL结构域和SLT结构域。高级结构分析发现,GzLysG蛋白以α螺旋和无规则卷曲结构为主,其分子表面有一条与溶菌酶活性密切相关的狭长裂缝。系统进化分析表明溶菌酶在物种进化过程中较为保守,且GzLysG与斑马鱼来源的溶菌酶表现出较近的亲缘关系。组织表达谱分析发现,GzLysG mRNA在免疫保护相关的皮肤、肌肉和鳃组织中具有较高表达量。结论GzLysG在扎那纹胸鮡固有免疫防御中可能发挥重要作用。

Differential gene expression profiling of gastric intraepithelial neoplasia and early-stage adenocarcinoma

AIM:To investigate the differentiated whole genome expression profiling of gastric high-and low-grade intraepithelial neoplasia and early-stage adenocarcinoma.METHODS:Gastric specimens from an upper magnifying chromoendoscopic targeted biopsy were collected from March 2010 to May 2013.Whole genome expression profiling was performed on 19 low-grade intraepithelial neoplasia(LGIN),20 high-grade intraepithelial neoplasia(HGIN),19 early-stage adenocarcinoma(EGC),and 19 chronic gastritis tissue samples using Agilent 4×44K Whole Human Genome microarrays.Differentially expressed genes between different types of lesions were identified using an unpaired t-test and corrected with the Benjamini and Hochberg false discovery rate algorithm.A gene ontology(GO)enrichment analysis was performed using the Gene Spring software GX 12.6.The differentially expressed gene was verified using a real-time TaqManPCR assay with independent tissue samples,including 26 LGIN,15 HGIN,14 EGC,and 20 chronic gastritis.The expression of G0S2 were further validated by immunohistochemical staining(IHC)in 24 LGIN,40 HGIN,30 EGC and 61 chronic gastritis specimens.RESULTS:The gene expression patterns of LGIN and HGIN tissues were distinct.There were 2521 significantly differentially expressed transcripts in HGIN,with951 upregulated and 1570 downregulated.A GO enrichment analysis demonstrated that the most striking overexpressed transcripts in HGIN compared with LGIN were in the category of metabolism,defense response,and nuclear factorκB(NF-κB)cascade.While the vast majority of transcripts had barely altered expression in HGIN and EGC tissues,only 38 transcripts were upregulated in EGC.A GO enrichment analysis revealed that the alterations of the immune response were most prominent in the progression from HGIN to EGC.It is worth noting that,compared with LGIN,289 transcriptswere expressed at higher levels both in HGIN and EGC.A characteristic gene,G0/G1 switch 2(G0S2)was one of the 289 transcripts and related to metabolism,the immune response,and the NF-κB cascade,and its expression was validated in independent samples through real-time TaqManPCR and immunohistochemical staining.In real-time PCR analysis,the expression of G0S2 was elevated both in HGIN and EGC compared with that in LGIN(P<0.01 and P<0.001,respectively).In IHC analysis,G0S2 immunoreactivity was detected in the cytoplasmic of neoplastic cells,but was undetectable in chronic gastritis cells.The G0S2 expression in HGIN was higher than that of LGIN(P=0.012,χ2=6.28)and EGC(P=0.008,χ2=6.94).CONCLUSION:A clear biological distinction between gastric high-and low-grade intraepithelial neoplasia was identified,and provides molecular evidence for clinical application.

呼吸道合胞病毒糖蛋白F和G在同一个重组痘苗病毒中的表达

在使用痘苗病毒启动子P11k、P25k和P7.5k研究呼吸道合胞病毒(RSV)糖蛋白F和G表达效率的基础上,构建成功同时表达BSV F和G糖蛋白的重组痘苗病毒(VMS11G75F)。结果表明,11k和7.5k启动子可以象单独表达RSV F或G一样有效地同时表达RSVF和G。荧光检测显示,RSV F和G主要分布于细胞膜表面,部分RSVF分布于胞浆近核膜处。这种荧光分布特征在RSV F和G单独表达和同时表达的重组痘苗病毒中无明显差别。Westernblot表明,痘苗病毒表达的RSV糖蛋白G具有一条分子量约为90kD的弥散带,与RSV感染细胞产生的RSV G相同;RSVF除具有与RSV感染细胞产生的F1(48kD)和F2(20kD)相同的两种形式外,尚有少量未被蛋白酶水解的F0(6skD)。RSVF和G同时表达的重组痘苗病毒VMS11G75F经传15代后,在表达水平和基因组水平上都具有良好的遗传稳定性。

开发EXPRESS数据模式的方法

随着ＳＴＥＰ标准应用的逐步广泛和深入，用ＥＸＰＲＥＳＳ语言描述的数据模式将广泛地应用在计算机集成制造系统（ＣＩＭＳ）中，因此，研究ＥＸＰＲＥＳＳ数据模式开发方法就具有十分重要的意义。本文提出了一种开发ＥＸＰＲＥＳＳ数据模式的具体方法。它综合了多种建立信息模型方法的特点，对ＩＤＥＦ１Ｘ的功能进行了扩充，增加了定义域约束（ＤｏｍａｉｎＣｏｎｓｔｒａｉｎｔ）的概念和实体关系断言的图形表示方法，有力地支持了ＥＸＰＲＥＳＳ数据模式的开发。

水稻谷蛋白GluA-2基因5′上游序列控制下的UidA基因在转基因水稻胚乳中的表达模式(英文)

为了研究谷蛋白胚乳特异性表达启动子在我国栽培稻品种中的表达模式 ,将UidA基因分别置于水稻谷蛋白GluA 2基因 750bp和 2 3kb上游序列下游 ,利用农杆菌转化法导入栽培稻品种中花 8号并获得转基因植株。Southernblot检测表明 ,UidA基因已经整合到水稻基因组当中并以单拷贝存在。Northernblot检测表明 ,开花后 13～15d和 11～ 13d ,UidA基因和水稻内源的GluA 2基因的表达量分别达到最高 ,随后逐渐降低。对转基因植株种子的GUS染色表明 ,UidA基因仅在胚乳中表达 ,在糊粉层中GUS表达量最高。测定了 2 3kb和 750bp转基因植株种子的GUS活性 ,结果表明前者的GUS活性是后者的 2～ 3倍。序列分析表明 ,位于GluA 2基因转录启始位点上游 2170bp的G

重组蛋白sHLA-G1的原核表达与纯化

为了进一步在蛋白质水平对人白细胞抗原HLA-G进行分析,利用已经构建好的克隆质粒pGEM-T-HLA-G1,进一步构建了重组原核表达质粒pET28a-sHLA-G1。SDS-PAGE检测表明,重组蛋白sHLA-G1在大肠杆菌(E.coli)中得到稳定表达,原核表达的重组蛋白sHLA-G1主要以包涵体形式存在。包涵体蛋白经充分洗涤、尿素溶解后用钴离子树脂纯化,并用SDS-PAGE和Western印迹进行了分析,鉴定结果表明,该蛋白纯度达到90%以上,并能与HLA-G特异性抗体87G反应。该研究结果为进一步的复性工作奠定了基础。

电离辐射诱导EL-4淋巴瘤细胞G_1期阻滞及相关蛋白的表达

探讨X射线诱导EL 1细胞G1期阻滞及相关蛋白的表达。采用PI荧光标记 ,流式细胞术检测细胞周期的变化。用单克隆抗体免疫荧光标记 ,流式细胞术检测蛋白表达的变化。结果表明 ,2 0Gy和 4 0Gy照射后 12 72h ,G1期EL 4细胞百分数显著高于假照射组 ( p <0 0 5- p <0 0 0 1)。 4 0Gy照射后EL 4细胞 p53蛋白表达从照射后 2h开始明显增高 ,持续至照射后2 4h ( p <0 0 5- p <0 0 0 1) ;p2 1蛋白表达在照射后 2h开始明显增高 ,持续至照射后 4 8h ( p <0 0 5- p <0 0 0 1) ;GADD4 5蛋白表达在照射后 2h开始明显增高 ,持续至照射后 4 8h ( p <0 0 5- p <0 0 0 1) ;MDM 2蛋白表达在照射后 4h开始明显增高 ,持续至照射后 2 4h ( p <0 0 5- p <0 0 0 1)。结果提示 ,中等剂量X射线照射可诱导EL 4细胞G1期阻滞。p53、p2 1和GADD4 5蛋白表达在电离辐射诱导EL

铅对胚胎大鼠海马神经元G蛋白mRNA表达的影响

目的研究铅对神经信号传导的关键部位──细胞膜上Ｇ蛋白（Ｇ－ｐｒｏｔｅｉｎｓ）的ｍＲＮＡ表达水平的影响。方法采用胚胎大鼠海马神经元无血清培养方法，加入s不同浓度的氯化铅溶液，设立无铅暴露的空白对照组，采用逆转录多聚酶链反应（ＲＴ－ＰＣＲ）技术半定量测定Ｇ蛋白三种不同类型α亚基，包括Ｇｓ、Ｇｉ２和Ｇｏｌα亚基ｍＲＮＡ表达水平，然后对结果进行相关矩阵分析。结果随着铅暴露水平的提高，Ｇｉ２α、Ｇｏ１αｍＲＮＡ表达水平显著增加，而ＧｓαｍＲＮＡ表达水平却无明显改变。结论铅暴露可干扰海马神经元Ｇ蛋白ｍＲＮＡ的表达，使Ｇ蛋白不同α亚基的ｍＲＮＡ表达水平失去平衡，从而影响神经信号的正常传递。

七鳃鳗g型溶菌酶的分子特征和抗菌活性

溶菌酶是先天免疫系统中对抗细菌病原体感染的一种关键蛋白.本研究从七鳃鳗中克隆g型溶菌酶基因.其酶基因c DNA为701 bp(Gen Bank序列号KP204854),开放阅读框为555 bp,编码由184个氨基酸组成的多肽,理论分子质量为20.24 k D,等电点为5.48,含有1个半胱氨酸残基,无信号肽.实时荧光定量PCR分析表明,七鳃鳗g型溶菌酶基因在各组织中广泛表达,其中在肠中表达量最高.脂多糖(LPS)体内刺激七鳃鳗后发现,溶菌酶在口腔腺和头肾表达量显著升高.以溶壁微球菌和哈维弧菌为底物检测重组g型溶菌酶的活性时,均表现出抗菌活性,最适p H为7.5,最适温度为35℃.扫描电镜分析表明,重组酶能够使溶壁微球菌破裂.以上结果均表明,g型溶菌酶在七鳃鳗的先天免疫系统防御病菌感染中起到重要作用.