Systemic delivery of human growth hormone by injection of genetically engineered myoblasts

A recombinant gene encoding human growth hormone (hGH) was stably introduced into cultured myoblasts with a retroviral vector. After injection of genetically engineered myoblasts into mouse muscle, hGH could be detected in serum for 3 months. The fate of injected myoblasts was assessed by coinfecting the cells with two retroviral vectors, one encoding hGH and the other encoding beta-galactosidase from Escherichia coli. These results provide evidence that myoblasts, which can fuse into preexisting multinucleated myofibers that are vascularized and innervated, may be advantageous as vehicles for systemic delivery of recombinant proteins.     

原核表达番鸭呼肠孤病毒σC与σB蛋白对雏番鸭的免疫保护

【目的】探讨原核表达的番鸭呼肠孤病毒（DRV）外壳蛋白σC与σB对于雏番鸭的免疫保护作用,为研制有效的DRV亚单位疫苗提供理论基础。【方法】对pET-30a-S3/BL21重组菌与pET-30a-S4 ORF2/BL21重组菌进行稳定性检验,表达的重组蛋白进行纯化和复性后,进行油乳剂制备,50只1日龄雏番鸭随机平均分为5组,即DRV σB蛋白免疫组、DRV σC蛋白免疫组、DRV σB + σC蛋白免疫组及PBS对照组和攻毒对照组,免疫组皮下接种剂量为500 μg/只,7日龄时进行二次免疫,14日龄时进行攻毒试验,ELISA和琼扩（AGP）检测抗体水平并计算保护指数。【结果】重组菌具有良好的稳定性,各免疫组的雏番鸭均在第2次免疫后7 d产生了一定效价的抗体,其中DRV σB + σC蛋白免疫组和DRV σC蛋白免疫组的雏番鸭血清抗体ELISA效价均为1﹕200,高于DRV σB蛋白免疫组的1﹕100。攻毒保护试验结果表明,联合免疫的保护指数最高,可达70,重组DRV σC保护指数次之,为60,重组DRV σB蛋白保护指数最低,仅为40。【结论】使用原核表达的DRV外壳蛋白σC与σB制备油乳剂,联合免疫可诱导雏番鸭快速产生一定效价的抗体,并提供良好的免疫保护力。     

基因工程菌表达效率对细菌总蛋白和包涵体中目的蛋白含量的影响

测定了猪生长激素基因工程菌不同表达效率时细菌总蛋白的含量以及包涵体中重组猪生长激素占包涵体总蛋白的比值。结果表明:随着表达效率的提高,细菌总蛋白含量缓慢增加,最后稳定在一定水平上;当表达效率增加,包涵体中重组猪生长激素占包涵体中总蛋白的百分数也增加,两者上升趋势基本一致。     

Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule

Human growth hormone (hGH) forms a 1:2 complex with the extracellular domain of its receptor-binding protein (hGHbp) as studied by crystallization, size exclusion chromatography, calorimetry, and a previously undescribed fluorescence quenching assay. These and other experiments with protein engineered variants of hGH have led to the identification of the binding determinants for two distinct but adjacent sites on hGH for the hGHbp, and the data indicated that there are two overlapping binding sites on the hGHbp for hGH. Furthermore, the binding of hGH to the hGHbp occurred sequentially; a first hGHbp molecule bound to site 1 on hGH and then a second hGHbp bound to site 2. Hormone-induced receptor dimerization is proposed to be relevant to the signal transduction mechanism for the hGH receptor and other related cytokine receptors.     

牛myf6基因真核表达载体的构建及在成肌细胞中的表达

【目的】构建牛myf6基因真核表达载体,并观察myf6真核表达载体转染鲁西黄牛成肌细胞后基因的表达和细胞形态的变化。【方法】在质粒pIRES2-EGFP的多克隆位点插入myf6基因构建真核表达载体pIRES2-EGFP-myf6,用脂质体技术转染鲁西黄牛成肌细胞,通过G418 筛选出稳定转染的细胞株。利用Western印记、Real-time PCR技术检测成肌细胞转染前后myf6基因、肌肉肌酸激酶基因和肌球蛋白轻链基因的表达量。【结果】与对照组相比,转染质粒的成肌细胞myf6蛋白和mRNA的表达量提高（P＜0.01）,肌肉肌酸激酶基因和肌球蛋白轻链基因的mRNA表达量提高（P＜0.01）。细胞形态观察显示成肌细胞融合为肌管。【结论】构建的真核表达载体pIRES2-EGFP-myf6能在成肌细胞中高效表达,myf6基因促进了成肌细胞向肌肉细胞分化。     

新城疫病毒F和HN核心片段在杆状病毒中的表达

为研制抗新城疫病毒的基因工程疫苗,将新城疫病毒F48E8株F和HN基因的核心片段克隆到杆状病毒转移载体pFastBacHTb中,然后转化到DH10Bac感受态细胞中与Bacmid杆状病毒穿梭载体进行转座重组,最后将重组子转染TN5细胞,得到含F片段和含F HN片段的重组杆状病毒rBac F和rBac F HN。PCR扩增结果证实F和F HN基因片段重组到杆状病毒基因组中;SDS-PAGE和Western blot检验结果表明F和F HN基因片段在重组病毒中得到了表达。     

Dimerization of human growth hormone by zinc

Size-exclusion chromatography and sedimentation equilbrium studies demonstrated that zinc ion (Zn2+) induced the dimerization of human growth hormone (hGH). Scatchard analysis of 65Zn2+ binding to hGH showed that two Zn2+ ions associate per dimer of hGH in a cooperative fashion. Cobalt (II) can substitute for Zn2+ in the hormone dimer and gives a visible spectrum characteristic of cobalt coordinated in a tetrahedral fashion by oxygen- and nitrogen-containing ligands. Replacement of potential Zn2+ ligands (His18, His21, and Glu174) in hGH with alanine weakened both Zn2+ binding and hGH dimer formation. The Zn(2+)-hGH dimer was more stable than monomeric hGH to denaturation in guanidine-HCl. Formation of a Zn(2+)-hGH dimeric complex may be important for storage of hGH in secretory granules.     

Vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D prevents latent herpes in mice

In humans, herpes simplex virus causes a primary infection and then often a latent ganglionic infection that persists for life. Because these latent infections can recur periodically, vaccines are needed that can protect against both primary and latent herpes simplex infections. Infectious vaccinia virus recombinants that contain the herpes simplex virus type 1 (HSV-1) glycoprotein D gene under control of defined early or late vaccinia virus promoters were constructed. Tissue culture cells infected with these recombinant viruses synthesized a glycosylated protein that had the same mass (60,000 daltons) as the glycoprotein D produced by HSV-1. Immunization of mice with one of these recombinant viruses by intradermal, subcutaneous, or intraperitoneal routes resulted in the production of antibodies that neutralized HSV-1 and protected the mice against subsequent lethal challenge with HSV-1 or HSV-2. Immunization with the recombinant virus also protected the majority of the mice against the development of a latent HSV-1 infection of the trigeminal ganglia. This is the first demonstration that a genetically engineered vaccine can prevent the development of latency.     

苏云金芽胞杆菌ICP基因工程菌的研究进展

综述了近年来国内外利用杀虫细菌苏云金杆菌ＩＣＰ基因构建工程菌，包括植物根际定居菌、植物疫苗、重组杀蚊蓝细菌和杆状病毒Ｂｔ重组菌等研究的历程及现状、成功的范例及存在问题．文中亦讨论了该研究领域的应用前景．     

TK基因重组缺陷山羊痘病毒构建及其生物学特性鉴定

[目的]利用同源重组技术构建TK基因缺陷的山羊痘病毒（GTPV）毒株,为研制出更安全、高效的GTPV弱毒疫苗和病毒载体提供备选毒株.[方法]采用PCR克隆GTPV AV41株TK基因（ORF56）及其侧翼基因组片段,在TK基因内部插入报告基因EGFP抗性基因gpt达盒,构建GTPV重组转移载体pTK-Eg.将重组转移载体pTK-Eg与GTPV AV41株共转染Vero细胞,通过空斑纯化筛选阳性重组病毒,鉴定其生长特性和遗传稳定性,并接种山羊评价其安全性和免疫原性.[结果]成功构建获得一株TK基因缺陷的重组病毒vTK-Eg.与亲本毒株GTPV AV41相比,vTK-Eg的生长特性及其形成的细胞病变均未发生显著改变,只是毒价略微下降100.5个数量级;在原代牛睾丸（BT）细胞上连续传代,至少在10代内保持遗传性状和病毒滴度稳定.接种vTK-Eg的山羊精神和食欲均正常,接种后体温升高和局部反应的严重程度均较接种亲本毒株GTPV AV41的山羊有所降低;vTK-Eg与GTPV AV41株诱导山羊产生的GTPV中和抗体水平无明显差异（P＞0.05）.[结论]构建的TK基因重组缺陷病毒vTK-Eg具有良好的遗传稳定性和免疫原性,较亲本毒株其安全性也有所提高,可作为研制GTPV基因工程弱毒疫苗和活载体疫苗的备选毒株.     

经基因工程改造的花生主要过敏原Arah2制备及其低致敏原性鉴定

构建经基因工程改造的Ara h 2表达载体,表达并纯化该蛋白,鉴定其过敏原性.将花生主要过敏原Arah2基因序列进行颠换,并将颠换后的序列进行合成,再将合成后的基因克隆到原核表达载体pET -32a(+)上,然后转入Origami宿主表达菌中;利用Isopropyl β-D -1 - Thiogalactopyrano...     

苏芸金杆菌MP-342IC-15工程菌构建

为了提高B.t.MP-342菌株对甜菜夜蛾的毒力,以B.t.MP-342菌株作亲株,根据MP-342菌株基因和毒蛋白的组成,采用重组DNA技术将对甜菜菜夜蛾高毒的CrylCa基因引入MP-342菌株,构建了工程菌B.t.MP-342IC-15工程菌.对该菌检测结果表明:MP-342IC-15工程菌表达两种Cry1类晶体蛋白,其分子量分别为135kD(CrylCa)和133kD(CrylAc),其CrylCa基因蛋白表达量占Cryl类晶体蛋白的35%和总晶体蛋白的29%.工程菌能良好表达CrylCa基因蛋白,且比例合适.据工程菌B.t.MP-342IC-15对甜菜夜蛾的生测表明.其毒力较亲株MP-342提高近1倍.对工程菌和亲株发酵上清液增效因子的增效活性进行了测定:工程菌上清液增效因子对小菜蛾具有一定增效作用;亲株上清液未显示对小菜蛾的增效作用.     

成肌细胞成脂过程中PPARγ、C/EBPα和Myogenin 基因启动子的甲基化变化

【目的】利用C2C12成肌细胞探讨肌源性干细胞成脂过程与调控成脂和成肌分化的关键转录因子PPARγ（peroxisome proliferator-activated receptor gamma）、C/EBPα（CCAAT/enhancer binding protein alpha）和Myogenin启动子区甲基化的关系。【方法】分别用2%马血清和三联诱导剂诱导C2C12细胞成肌和成脂分化,在马血清促进的成肌分化第0、1、3和5天收集细胞进行姬姆萨染色观察肌管形成情况;在三联诱导的成脂分化第0、2、4、6和10天收集细胞进行油红O染色观察脂滴形成情况;提取成肌诱导第0、1、3、5天和成脂诱导第0、2、4、6天的RNA和DNA,分别采用qRT-PCR检测成肌和成脂分化相关基因的表达,采用重亚硫酸盐测序的方法检测PPARγ、C/EBPα和Myogenin启动子区甲基化的变化,并分析基因表达与甲基化状态的相关关系。【结果】①C2C12细胞经马血清诱导形成了多核肌管,表达成肌相关基因,但不表达脂肪特异性基因;三联诱导使C2C12细胞自主分化的肌管中沉积了脂滴,同时表达成脂和成肌相关基因;②重亚硫酸盐测序结果表明,在未分化的成肌细胞中,PPARγ基因启动子的甲基化水平是61%,在三联诱导第2、4和6天,其甲基化程度依次为49%、39%和42%,呈逐渐去甲基化趋势,与PPARγ基因转录负相关;在马血清诱导第3和5天,甲基化水平为56%和48%,与未分化的成肌细胞相比差异不显著,同时PPARγ基因的表达水平也没有显著变化;③Myogenin基因在马血清促进的成肌过程中甲基化水平不断降低（49%、42%、35%和34%）,转录水平急剧增加;在三联诱导过程中,Myogenin启动子的甲基化水平由0天的49%下降为第1天的37%、第3天的41%和第5天的38%,但下降幅度弱于马血清诱导的成肌过程,这一结果与降低的Myogenin转录上调相吻合;④C/EBPα基因在未分化的C2C12细胞中呈低甲基化状态,甲基化程度仅为1.6%,在成肌分化和脂肪沉积过程中均未发生显著变化,与基因表达无显著关联。【结论】成脂和成肌关键转录因子PPARγ和Myogenin启动子区的DNA甲基化变化参与了成肌细胞的分化和脂肪沉积的调控。     

High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis

A strategy, called alanine-scanning mutagenesis, was used to identify specific side chains in human growth hormone (hGH) that strongly modulate binding to the hGH receptor cloned from human liver. Single alanine mutations (62 in total) were introduced at every residue contained within the three discontinuous segments of hGH (residues 2 to 19, 54 to 74, and 167 to 191) that have been implicated in receptor recognition. The alanine scan revealed a cluster of a dozen large side chains that when mutated to alanine each showed more than a four times lower binding affinity to the hGH receptor. Many of these residues that promote binding to the hGH receptor are altered in homologs of hGH (such as placental lactogens and prolactins) that do not bind tightly to the hGH receptor. The overall folding of these mutant proteins was indistinguishable from that of the wild-type hGH, as determined by strong cross-reactivities with seven different conformationally sensitive monoclonal antibodies. The alanine scan also identified at least one side chain, Glu174, that hindered binding because when it was mutated to alanine the receptor affinity increased by more than a factor of four.     

羊传染性脓疱病毒B2L基因片段重组腺病毒载体的构建（摘要）（英文）

[目的]利用腺病毒载体系统构建羊传染性脓疱病毒B2L基因重组腺病毒载体。[方法]以从羊传染性脓疱病毒株JLSY04中提取的基因组DNA为模板,PCR扩增获得B2L目的基因片段;然后将B2L目的基因克隆至PD-NR-CMV载体,筛选阳性克隆获得质粒CTC572-6;再将质粒CTC572-6与腺病毒载体进行同源重组,筛选阳性克隆,并进行菌液PCR、酶切、测序等鉴定。[结果]经酶切和基因测序等鉴定,成功构建了携带羊传染性脓疱病毒B2L基因的重组腺病毒载体CTC572Ade-30。[结论]为羊传染性脓疱基因工程疫苗的进一步研究奠定基础。     

羊传染性脓疱病毒B2L基因片段重组腺病毒载体的构建

[目的]利用腺病毒载体系统构建羊传染性脓疱病毒B2L基因重组腺病毒载体。[方法]以从羊传染性脓疱病毒株JLSY04中提取的基因组DNA为模板,PCR扩增获得B2L目的基因片段;然后将B2L目的基因克隆至PDNR-CMV载体,筛选阳性克隆获得质粒CTC572-6;再将质粒CTC572-6与腺病毒载体进行同源重组,筛选阳性克隆,并进行菌液PCR、酶切、测序等鉴定。[结果]经酶切和基因测序等鉴定,成功构建了携带羊传染性脓疱病毒B2L基因的重组腺病毒载体CTC572Ade-30。[结论]为羊传染性脓疱基因工程疫苗的进一步研究奠定基础。     

Protection from genital herpes simplex virus type 2 infection by vaccination with cloned type 1 glycoprotein D

Guinea pigs were vaccinated with truncated herpes simplex virus type-1 (HSV-1) glycoprotein D produced in the genetically engineered mammalian cell line gD10.2. Vaccinated animals formed antibodies that neutralized both HSV-1 and herpes simplex virus type 2 (HSV-2) in an in vitro neutralization assay. Vaccinated animals were challenged with HSV-2 by intravaginal infection. Animals that received the immunogen in Freund's complete adjuvant were completely protected from the clinical manifestations of genital HSV-2 infection. Animals that received the immunogen incorporated in alum adjuvants were partly protected from clinical disease; the infections that did develop were significantly less severe than those that occurred in control animals injected with adjuvant alone. The results demonstrate that immunization with a purified viral protein can provide significant protection against primary genital infection by HSV-2 in guinea pigs.     

通过耐酸性改造在昆虫细胞中组装FMDV空衣壳结构

 【目的】口蹄疫病毒（FMDV）对酸很敏感,当pH值低于7时,二十面体对称的衣壳结构就会裂解成12S的五聚体。而昆虫细胞培养基的正常pH值在6.3左右,很难应用杆状病毒表达系统在昆虫细胞中组装天然的FMDV空衣壳结构。本研究旨在通过耐酸性改造,应用杆状病毒表达载体在昆虫细胞中组装产生AsiaⅠ型FMDV空衣壳结构。【方法】应用定点突变技术改变VP3上的H140和H143为亮氨酸,以提高空衣壳对酸的耐受性。将改造和未改造的P12A基因和3C基因插入带双启动子的杆状病毒转移载体pFastBacTM Dual 中,通过在大肠杆菌内转座重组,获得重组杆粒,转染Sf 9细胞,获得两株表达FMDV全衣壳蛋白的重组杆状病毒Bac mP12A3C和Bac P12A3C。重组杆状病毒经增殖后感染High FiveTM细胞,进行目的蛋白的表达。【结果】通过Western blotting检测表明目的基因均获得表达,且衣壳蛋白被3C蛋白酶成功地加工裂解。双抗体夹心ELISA和免疫荧光检测结果表明表达蛋白主要集中在细胞膜上,且具有很好的抗原性。通过电镜观察到P12A基因改造的重组杆状病毒在昆虫细胞内产生了直径为25～30 nm的空衣壳结构,而P12A基因未改造的重组杆状病毒观察到很多直径小得多的结构。【结论】本研究首次用电子显微镜在昆虫细胞中观察到FMDV完整的空衣壳结构,为基因工程亚单位疫苗和新型诊断试剂的研发奠定了基础。     

苏云金芽孢杆菌cry1Ea基因的克隆及 生物信息学分析

不同来源的苏云金芽孢杆菌能产生多种多样的晶体(Cry)蛋白.基于这个特性,人们可以通过基因工程的手段向工程菌中转入编码多种Cry毒素的基因来控制虫害.通过DNA重组技术,从BtHZM2菌株中克隆出了cry1Ea基因,对其进行了生物信息学分析,同源比对结果表明.cry1Ea8基因的核苷酸序列与已知cry1Ea的同源性为99.77%～99.91%.对应的氨基酸序列同源性为99.49%～99.74%.对cry1Ea8基因的分析还揭示出了cry1Ea8及其编码蛋白的一些生物和理化性质.结构域预测表明,Cry1Ea8由3个结构域组成,其中N-末端螺旋状结构域与膜插入与孔隙形成有关,而第二和第三个结构域与受体的结合有关.该研究为转基因抗虫植物和微生物杀虫工程菌的构建提供了新的基因来源.     

水稻纤维素合酶多克隆抗体的制备和鉴定

纤维素合酶是参与纤维素β-1,4-葡聚糖链延伸的主要催化亚基,为进一步研究水稻中纤维素合酶的作用机理,采用DNA重组技术,将纤维素合酶基因CesA1、CesA2、CesA3克隆入表达载体pGEX-4T-3,构建重组质粒.在大肠杆菌JM109中,经异丙基硫代半乳糖苷(IPTG)诱导后在原核细胞中成功表达了3种纤维素合酶的...