Cloning of rainbow trout (Oncorhynchus mykiss) histone H3 promoter and the activity analysis in rare minnow (Gobiocypris rarus)

Rainbow trout historic H3 (RH3) promoter was cloned via high fidelity PCR. The cloned RH3 promoter was inserted into a promoter-lacked vector pEGFP-1, resulting in an expression vector pRH3FGFP-1. The linearized pRH3EGFP-1 was microinjected into fertilized eggs of rare minnows and the sequential embryogenetic processes were monitored under a fluorescent microscope. Strong green fluorescence was ubiquitously observed at as early as the gastrula stage and then in various tissues at the fry stage. The results indicate that RH3 promoter, as a piscine promoter, could serve in producing transgenic Cyprinoid such as rare minnow. Promoter activity of RH3, CMV and common carp beta-actin (CA) were compared in rare minnow by the expression of respective recombinant EGFP vectors. The expression of pCMVEGFP occurred earlier than the following one, pRH3EGFP-1, and then pCAEGFP during the embryogenesis of the transgenics. Their expression activities demonstrated that the CMV promoter is the strongest one, followed by the CA and then the RH3.

Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome

Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV. (C) 2003 Elsevier B.V. All rights reserved.

Measurement of estrogen activity in two Chinese lakes

Estrogenic activities of samples from two Chinese lakes, Ya-Er Lake and Donghu Lake, were measured by in vitro recombinant yeast assay and found in both lakes polluted with industrial and domestic wastewater. In methanol extracts of lake water samples the estrogen-like activity was higher than in toluene extracts. These results have been inverted for solid samples. Furthermore, the EC50 value of the water samples was close to the original concentration in the lake.

Growth hormone gene transfer in common carp

The first successful case of transgenic fish was achieved in 1984. It is in a model system that the integration and expression of recombinant human growth hormone (hGH) in host red common carp (Cyprinus carpio, red var.) have been thoroughly studied. Recently, the integration sites have been recovered and characterized. Compared with non-transgenic peers, hGH-transgenic fish are prior in dietary utilization and growth performance. In view of bio-safety and bio-ethics, an "all-fish" construct CAgcGH, grass carp growth hormone fused with common carp P-actin promoter, has been generated and transferred into Yellow River carp (C carpio, local strain in Yellow River) fertilized eggs. Under middle-scale trial, CAgcGH-transgenics show higher growth rate and food conversion efficiency than the controls, which is consistent to laboratory findings. To avoid the potential impact of transgenic fish on the environment, a sterile strain of transgenic triploid fish has been successfully produced. The "all-fish" transgenic common carp is also approved safe enough as daily food, according to a test based on the pathological principles of new medicines issued by the Ministry of Health of China. The "all-fish" transgenic common carp with growth enhancement is now ready for market, but looking for governmental authorization. (C) 2003 Editions scientifiques et medicales Elsevier SAS and Ifremer/IRD/Inra/Cemagref. All rights reserved.

Embryonic and genetic manipulation in fish

Fishes, the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics. Nuclear transplantation in fish has been thoroughly studied in China since 1960s. Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults. Most importantly, nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish. This was the first case of cloned fish with somatic cells. Based on the technique of microinjection, recombinant MThGH gene has been transferred into fish eggs and the first batch of transgenic fish were produced in 1984. The behavior of foreign gene was characterized and the onset of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis. This eventually led to the transgenic mosaicism. The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults. The transgenic common carp were more efficient in utilizing dietary protein than the controls. An "all-fish" gene construct CAgcGH has been made by splicing the common carp beta-actin gene (CA) promoter onto the grass carp growth hormone gene (gcGH) coding sequence. The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait. Combination of techniques of fish cell culture, gene transformation with cultured cells and nuclear transplantation should be able to generate homogeneous strain of valuable transgenic fish to fulfil human requirement in 21(st) century.

Estradiol stimulates growth hormone production in female goldfish

The effects of estradiol (E(2)) on growth hormone (GH) production was investigated in gonad-intact female goldfish. It was first necessary to generate a specific antibody for use in immunocytochemistry, Western, and dot-blot analyses of GH production. To accomplish this, grass carp GH (gcGH) cDNA was cloned by the reverse transcription polymerase chain reaction (RT-PCR) and expressed in Echerichia coli and a specific polyclonal antibody to recombinant gcGH was generated in the rabbit. In Western blot, the anti-gcGH antibody specifically immunoreacted with recombinant gcGH, purified natural common carp GH, and with a single 21.5-kDa GH form from pituitary extracts of grass carp, common carp, goldfish, and zebrafish but not salmon, trout, or tilapia. Intraperitoneal injection of the recombinant gcGH enhanced the growth rates of juvenile common carp demonstrating biological activity of this GH preparation. Electron microscopic studies showed that the anti-gcGH-I antibody specifically reacted with GH localized in the secretory granules of the goldfish somatotroph. Using anti-gcGH-I in a dot-blot assay, it was found that in vivo implantation of solid silastic pellets containing E(2) (100 mu g/g body weight for 5 days) increased pituitary GH content by 150% in female goldfish. In a second, independent study employing a previously characterized anticommon carp GH antibody for radioimmunoassay, it was found that E(2) increased pituitary GH content by 170% and serum GH levels by approximately 350%. The E(2)-induced hypersecretion of GH and increase in pituitary GH levels was not associated with changes in steady-state pituitary GH mRNA levels, suggesting that this sex steroid may enhance GH synthesis at the posttranscriptional or translational level. Previous observations indicate that GH can stimulate ovarian E(2) production. The present results show that E(2) can in turn stimulate GH production, indicating the existence of a novel pituitary GH-ovarian feedback system in goldfish. (C) 1997 Academic Press.

HORMONAL REPLACEMENT THERAPY IN FISH - HUMAN GROWTH-HORMONE GENE-FUNCTION IN HYPOPHYSECTOMIZED CARP

Transgenic common carp, Cyprinus carpio, produced by the microinjection of fertilized eggs with a linearized chimeric plasmid pMThGH, a human growth hormone (hGH) gene with a mouse metallothionein-I (MT) gene promoter in pBR322, were used to produce F1 and F2 transgenics. Following hypophysectomy of the transgenic F2 common carp, non-transgenic common carp and non-transgenic crucian carp, growth was monitored for up to 110 days. In addition, recombinant hGH was injected subcutaenously into a group of the non-transgenic crucian carp. Growth rate analyses indicated that (1) hypophysectomy of non-transgenic common carp and crucian carp results in the cessation of growth, (2) hGH administration can stimulate the growth of hypophysectomized crucian carp and (3) hypophysectomized hGH-transgenic common carp continue to grow in the absence of their own growth hormone, suggesting that the hGH-transgene is being expressed in tissues other than the pituitary.

重组人内皮抑素的基因表达与表征

内皮抑素具有抑制内皮细胞增殖和抗肿瘤新生血管生成活性，在小鼠实验中对多种肿瘤取得了有效的结果，有望成为一种新型抗肿瘤药物。本论文从基因的获取到在大肠杆菌中的高效表达，系统地研究了重组内皮抑素的全过程，并对其体内、体外的生物学活性及多种光谱学性质进行了深入的研究。采用Trizol试剂改进法从人胚肝组织中制备细胞mR NA，通过RT-PCR方法获得人内皮抑素基因。应用基因工程技术构建了亚克隆载体和表达载体，在大肠杆菌BL21（DE3）中以包涵体方式表达出N-末端带有6XHisTag标签的融合蛋白，表达量最多达到菌体总蛋白的42％。建立了包涵体蛋白的复性和纯化方法，得到具有较高纯度的重组内皮抑素蛋白。首次用质谱法证实从大肠杆菌的包涵体中获得的重组蛋白，因宿主菌中水解甲硫氨酸的酶不能对所有包涵体蛋白起作用，而在目的蛋白中会棍有N-末端具有甲硫氨酸的产物。对内皮抑素基因做定点突变，首次将内皮抑素蛋白的N-末端锌离子结合位点的的His2、His4突变成Leu2、Va14，构建了点突变的亚克隆载体，为以后研究其作用机理提供了条件。重组内皮抑素能够抑制鸡胚尿囊膜血管生成和人脐静脉内皮细胞ECV-304的增殖，在小鼠体内实验中对黑色素瘤产生良好的抑瘤效果（P＜0.01）。通过细胞周期分析，首次发现重组内皮抑素在G2期抑制 ECV-304细胞的增殖。电镜观察发现受内皮抑素作用的ECV-304细胞，产生了凋亡小体并出现多种细胞凋亡特征。同时，首次发现内皮抑素能使Balb／c 3T3细胞产生凋亡。计算机结构模拟结果表明重组内皮抑素蛋白与天然蛋白具有相似的空间结构。研究了内皮抑素与锌离子、肝素、稀土离子作用后蛋白质的光谱学性质和二级结构的变化，首次发现偏酸性pH对肝素与内皮抑素的结合作用有特殊影响，为研究内皮抑素特异抑制肿瘤组织新生血管的生成提供了实验依据。从人胚肝组织出发初步完成了人硒蛋白P亚克隆载体的构建。

PAR4 和TFF2 在胃肠癌中的表达变异与临床关系以及 TFF2 的重组表达

胃癌和肠癌是常见的威胁人类健康的消化道恶性肿瘤，其发生发展涉及多因 子的作用及调控。其中，在胃肠道都有表达的蛋白酶激活受体（PARs）和三叶 因子蛋白（TFFs）家族都参与肿瘤发生发展的调控过程。正常生理条件下，PARs 的表达与胃肠道消化液的分泌和肌肉的收缩舒张相关。同时，在胃肠道肿瘤的发 生、浸润和转移过程中PARs和TFF2也发挥了作用。而PAR-4，除了具有凝血酶 激活后的血小板聚集功能外，还参与感染、细胞迁移和肿瘤的发生发展。在溃疡 性结肠炎，肠癌组织以及某些肠癌细胞系中都出现PAR4的异常表达，而这种异 常表达可能作为启动肠癌发生的重要环节。TFFs家族蛋白能够对抗粘膜损伤并 且参与修复以发挥保护胃肠道的功能。在肿瘤发生中，三叶因子既有报道作为肿 瘤抑制因子，又有报道作为潜在的肿瘤促进因子。含两个三叶因子结构域的 TFF2，主要表达在胃粘膜的颈细胞。在胃溃疡、慢性萎缩性胃炎及胃癌中，TFF2 的表达具有下降的趋势；而且分化程度越低的胃癌，TFF2的表达量越少，这是 因为TFF2的表达与胃粘膜细胞的增殖和恶性转移相关。在肠道，TFF2可以抑制 一氧化氮（NO）的生成以调节由NO引起的肠炎；在肠炎老鼠的模型中，TFF2 能减轻炎症和溃疡发生的程度，表明TFF2可能通过调节机体的免疫反应来抑制 肠道炎症的发生。 而本实验室前期对大蹼铃蟾皮肤分泌物中获得的新型血小板激动蛋白 -Bm-TFF2与PARs相互作用的实验，促使我们去研究人TFF2与PARs的关系。由 于免疫组化提示TFF2和PAR4在正常胃黏膜中都分布在从基底部到中间的位置， 而且TFF2第二个Loop区序列的保守性，以及和PAR4连接配体（tethered-ligand） 的高度相似性，促使我们推测PAR4和TFF2之间是否存在一种相互作用，或者 hTFF2是否能调节PAR4的生物学活性。所以该篇论文落脚于PAR4和hTFF2，着 重介绍PAR4和TFF2在胃肠道肿瘤中的表达变异以及TFF2对过表达PAR4的细胞 的趋化作用。 我们先用半定量PCR方法检测TFF2和PAR4在胃癌、肠癌及周围远癌部位组 织中mRNA的表达水平。结果提示两个基因在胃癌组织中的表达较周围远癌部位组织减弱，而在肠癌组织中的表达则较周围远癌部位组织增强。Western blotting 也得到相似的结果。为进一步明确PAR4和TFF2在胃癌和肠癌中表达的具体变化 情况，我们继而用实时荧光定量PCR对28例胃癌和38例肠癌及其周围远癌部位组 织中TFF2和PAR4的表达进行了研究。结果显示胃癌组织中两个基因mRNA的表 达都显著低于远癌部位组织（P<0.001），而肠癌组织中两个基因mRNA的表达 则显著高于远癌部位组织（P<0.001）。结合临床病理资料提示PAR4在淋巴结转 移的胃癌患者中的表达低于无淋巴结转移的患者（P<0.05），在胃窦癌中的表达 明显低于非胃窦癌（P<0.05）；而发生淋巴结转移的肠癌患者其TFF2和PAR4基 因的表达都显著高于无淋巴结转移的肠癌患者（P<0.05）；两个基因在中低分化 肠癌中的表达也显著高于高分化肠癌（P<0.001）。免疫组化结果也提示TFF2和 PAR4在胃癌中的表达显著低于周围远癌部位组织（P<0.001），而在肠癌中的表 达则显著高于周围远癌部位组织（P<0.001）。表明TFF2和PAR4在胃肠道肿瘤的 发生中可能受到某些因素的调节而协调性地一致性表达。 在细胞水平上，我们发现在同等浓度hTFF2的诱导下，过表达PAR4的Lovo 稳定株的细胞迁移能力较不表达者明显增强，并且hTFF2的促细胞迁移活性呈剂 量依赖性，同时伴随ERK1/2磷酸化的增强。同时，过表达PAR4的Lovo细胞增殖 能力强于无PAR4表达的细胞，但TFF2作用后其增强能力反而下降，表明TFF2 对过表达了PAR4的Lovo细胞具有抗增殖的能力。 总之这些结果提示PAR4和TFF2在胃肠道中协同表达的现实为两者之间产 生一定的作用提供了基础，而且这种共存为粘膜受损后的修复，组织自身平衡状 态的维持都发挥了一定的作用，同时也为临床相关疾病的诊断，治疗及预后提供 一个新的理论依据。当然，生理和病理情况下，存在于PAR4和TFF2之间的调控 和相互作用的分子机制仍不清楚，这也是进一步研究的关键所在。 为探讨其它动物体内三叶因子家族蛋白结构和功能的关系，我们进而利用原 核表达体系构建并表达纯化了Bm-TFF2以及它的两个单结构域。由于Bm-TFF2 分子中有三对二硫键，所以我们选用pET-32a表达体系表达融合的重组蛋白，并 利用融合蛋白N端引入的Xa因子酶切位点将融合蛋白中的硫氧还蛋白切除，亲和 柱及反向高压液相色谱纯化游离的重组蛋白。重组的Bm-TFF2全长具有血小板聚集活性，而第一个结构域只有诱导血小板变形的作用；三种重组蛋白都具有剂量 依赖性地诱导AGS细胞迁移的功能，但三种重组蛋白的细胞迁移活性无明显差 异。pET-32a表达体系成功表达Bm-TFF2的事实为我们研究人三叶因子家族蛋白 结构及功能关系提供一种方便而可靠的手段。

重组腺病毒载体vAd2tat 的构建及其在细胞中的表达

　目的　构建含HIV21 tat 基因重组腺病毒,观察在不同细胞中外源蛋白Tat 的表达,作为DC 抗HIV 疫苗的基 础。方法　通过PCR 扩增,获得HXB2 tat 的cDNA 片段,定向克隆入腺病毒转移载体pTrack2CMV ,线性化后转化含有腺病毒骨 架pAd2easy21 的大肠杆菌BJ5183 ,获得同源重组的质粒prAd2tat , Pac Ⅰ酶切纯化后转染293 细胞,包装成具有感染力的复制缺 陷型重组腺病毒vAd2tat 。结果　经PCR、酶切及DNA 序列测定,插入片段大小、方向正确,获得具有感染力的含有HIV21 tat 基 因的重组腺病毒;通过Western blot 方法检测,重组腺病毒在293 细胞中表达出Mr 为15 000 的蛋白。结论　成功构建了含有 HIV21 tat 基因的腺病毒,并观察到该基因在细胞中的表达。

HIV-1 C亚型gp120重组腺病毒载体的构建及gp120负载人树突状细胞疫苗的初步研究

获得性免疫缺陷综合征(AIDS)是一种由人类免疫缺陷病毒(HIV)引起的，以全身免疫系统受到严重损害为特征的传染性疾病。从目前HIV-1的流行趋势来看，HIV-1 C亚型已经成为全球最主要的流行株之一，因此，针对HIV-1 C亚型的疫苗设计颇为重要。gp120作为HIV-1的包膜糖蛋白，能够诱导广泛的中和抗体反应，中和进入机体的病毒粒子，阻止病毒早期感染，所以本实验选取HIV-1 C亚型密码子优化的gp120作为免疫原进行研究。目前的疫苗研究中，腺病毒载体是较理想的病毒载体之一，具有安全性好、外源基因容纳量大、感染效率高、操作简便等优点。我们以复制缺陷型腺病毒为载体，构建了表达HIV-1 C亚型密码子优化的gp120的重组腺病毒vAd-gp120，经Western Blot方法检测到了gp120蛋白的表达。树突状细胞（DC）是已知最强的抗原呈递细胞(APC)，也是目前发现的唯一能够刺激初始型T细胞增殖的细胞。经抗原致敏的DC可通过MHC-Ⅰ、MHC-Ⅱ途径递呈抗原，并激活T细胞，从而激发体内的体液免疫和特异性细胞免疫反应。我们利用Amaxa系统将HIV-1 C亚型gp120基因转入人外周血单核细胞来源的DC，构建了以DC为载体的治疗性疫苗，并对其功能进行初步研究，发现负载gp120的DC能够显著刺激淋巴细胞的增殖、增强CD8T细胞表面活化分子CD25的表达以及促进CD8T细胞分泌++IFN-γ，为下一步DC治疗性疫苗的体内研究奠定了基础。

Genetic analysis of all-fish growth hormone gene transferred carp (Cyprinus carpio L.) and its F-1 generation

Recombinant "all-fish" growth hormone gene (GH) was microinjected Into the fertilized eggs of carp. A comparison between the growth traits of transgenics and non-transgenics was carried out, and the transgenic individuals with significant "fast-growing" effect were successfully gained. A comparison on the reproductivities was also given out between the transgenics and their non-transgenic siblings, and showed that the reproductive capacity of transgenics was substantially equivalent to those of the non-transgenics. On the other hand, the genetic separation and the characteristic distribution of the F-1 generation were genetically analyzed, which gave solid evidence for the hypothesis that 2-3 chromosomes are integrated with transgene. In addition, the distinct biological effects for multisite-integrated transgenes were further discussed. The present study opens a door for the breeding of "fast-growing" transgenic fish.

抗黑色素瘤ScFv-超抗原SEA重组免疫毒素的构建与表达

提取鼠抗人黑色素瘤杂交瘤细胞株HB8759的总RNA，反转录成cDNA，用抗体可变区混合引物扩增出全套重、轻链可变区基因（VH、VLDNA），通过（Gly4Ser）3连接肤基因把VH和VL基因装配成单链抗体（ScFv）基因，将其克隆到噬菌粒载体pCANTABSE中，构建单链抗体噬菌体抗体库。用LIBr黑色素瘤细胞对抗体库进行了3轮亲合筛选。随机挑选克隆进行hage-ELISA鉴定，结果获得了2株具有较高ELISA活性的噬菌体单链抗体。序列测定证实得到的ScFv符合抗体可变区的结构特点，与已发表的鼠抗体可变区基因有较高的同源性。采用酶切连接和重叠PCR连接两种方法将抗黑色素瘤单链抗体基因和去除N端信号肤的金黄色葡萄球菌肠毒素A（SEA）基因进行融合，并将融合基因克隆于pET28-a表达载体的H招标签下游。SDS-PAGE分析表明，两种构建方法均表达了相对分子量约SOkD的蛋白条带，与预期目的蛋白分子量相符，主要以包涵体的形式存在，表达量占菌体蛋白的27％。将重组质粒在大肠杆菌中诱导表达，用盐酸肌溶解包涵体，Ni-NTA鳌合层析柱一步法纯化包涵体，再通过透析使目的蛋白复性。凝胶灰度扫描显示蛋白纯度达90％，凝胶电泳呈单一条带，蛋白量达0.47mg／mL。用健康人外周血单个核细胞作为效应细胞，LDH法检测ScFv-SEA融合蛋白对LiBr黑色素瘤细胞、MCF7乳腺癌的体外抑制率。结果表明该融合蛋白可以通过活化效应细胞，对表达相关抗原的肿瘤细胞产生有效的抗增殖作用，而对不表达该抗原的肿瘤细胞作用不显著。说明该融合蛋白赋予了SEA抗黑色素瘤特异性。以上实验结果为我们研究SEA对黑色素瘤的靶向杀伤作用奠定了可靠的实验基础。