牛生长激素cDNA的分子克隆

我们克隆了与牛生长激素Poly(A)⁺RNA互补的DNA(cDNA)。首先从小牛垂体中提纯总的Pply(A)⁺RNA,用AMV逆转录酶合成单链cDNA,以单链cDNA为模板合成双链cDNA,用多聚G及多聚c谱尾法将双链cDNA克隆到pBR322质粒的Pst I位点上,构建成牛垂体PoIy(A)⁺RNA的cDNA文库。以牛生长激素基因为探针,筛选出7个阳性菌落,经电泳鉴定有两个菌落(1号和2号)含有大于500bp的插入片段。1号克隆经酶切图谱、southeTn blot 杂交及序列分析证实含有牛生长激素的编码序列。     

抗牛生长激素单克隆抗体及其免疫亲和吸附柱的制备

我们用杂交瘤技术获得了分秘抗牛生长激素单克隆抗体的细胞系(4B-2)。接种此细胞于小鼠所产生腹水的抗体含量达10mg／ml。经免疫亲和层析方法纯化的单克隆抗体,属1BG1型。将单克隆抗体偶联到Sepharose 4B上,创成了免疫亲和吸附柱,可以从牛垂体匀浆中一步纯化牛生长激素。偶联有50mg单克隆抗体的亲和柱一次可结合3mg牛生长激素。经单克隆抗体亲和层析纯化的牛生长激豢,保持了与兔肝细胞膜受体结合的能力,及在去垂体大白鼠中促进胫骨生长板生长的功能。     

牛生长激素基因在马铃薯中的表达

将牛生长激素基因ｃＤＮＡ 与Ｐａｔａｔｉｎ ＣｌａｓｓＩ启动子及ＮＯＳ３终止子连接,构建了表达载体ｐＰＢＧＴ． 用直接法将表达载体转入农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍ ｅｆａｃｉｅｎｓ） ＬＢＡ４４０４（ｐＲＡＬ４４０４）菌株, 用此菌株转化马铃薯（Ｓｏｌａｎｕｍ ｔｕｂｅｒｏｓｕｍ ）得到再生植株． 经ＮＰＴⅡ活性检测,总ＤＮＡ ＰＣＲ和Ｓｏｕｔｈｅｒｎ ｂｌｏｔ证明目的基因已整合到马铃薯基因组中．ＲＮＡ 点杂交和Ｗｅｓｔｅｒｎ ｂｌｏｔ表明牛生长激素基因已在转基因马铃薯块茎中转录和表达     

牛生长激素基因的人工合成,重组克隆及高效表达

本文通过设计选择大肠杆菌高频利用密码子代替天然密码子,人工全合成３２个寡聚核苷酸片段;连接并克隆获得Ａ（２７８ｂｐ）、Ｂ（８８ｂｐ）、Ｃ（２２４ｂｐ）３个较大ＤＮＡ片段;经重组克隆、定位突变等获得阳性克隆;双向ＤＮＡ序列测定分析表明获得了两种人工全合成牛生长激素（ｂＧＨ）编码基因,即编码Ｎ－Ｍｅｔ－Ａｌａ－ｂＧＨ和Ｎ－Ｍｅｔ－Ｐｈｅ－ｂＧＨ的两个基因,而至今尚未见有人工全合成ｂＧＨ基因的报道。构建了在ＰＬｐｒｏｍｏｔｅｒ控制下含上述合成基因的表达质位ｐＢＬｂＧＨＥ７Ａ１和ｐＢＬｂＧＨＥ８,并在大肠杆菌中进行了温敏诱导表达。经ＳＤＳ－ＰＡＧＥ分析,表达产物占全菌蛋白的３７％以上。Ｗｅｓｔｅｒｎ－ｂｌｏｔ分析和纯化产物的Ｎ端氨基酸序列测定结果都表明上述两种人工全合成ｂＧＨ基因在大肠杆菌中得到了高效表达,表达量为现今国际文献报道的高限水平。     

哺乳动物细胞中基因工程人生长激素的提纯和性质研究

 在哺乳动物细胞中克隆表达的人生长激素,经等电点和乙醇沉淀后,再用亲和层析法,获得产品。经对该产品的各项生化指标分析鉴定和活力测定,所得结果都与天然人生长激素相符合,证明其性质相同。     

用原位分子杂交技术定位牛生长激素基因于5号染色体

本工作利用放射性标记的ｂＧＨ基因（３．０ｋｂ）为探针,通过原位杂交定位牛生长激素基因于染色体５ｑ２２－２６内。该结果与以前的ｂＧＨ基因定位的结果不同,讨论了基因探针、基因定位方法等方面与定位准确性的关系。     

Expression of the Bovine Growth Hormone Alters the Root Morphology in Transgenic Tobacco Plants

The bovine growth hormone (bGH) is a natural peptide hormone that controls the differentiation, growth and metabolism, and is produced in the pituitary gland of cows. For the production of bGH from plants, two different bgh clones, of which the pGAbGH1 contaions only mature peptide sequences and the pGAbGH15 contains signal sequences and the first intron, as well as mature peptide sequences, were used. Those bghs under the control of the CaMV 35S promoter and NOS terminator were introduced to tobacco plants via Agrobacterium tumefaciens-mediated transformation. By PCR analyses using bgh and nptII specific primers, 17 and 21 putative transformants were respectively selected from pGAbGH1- and pGAbGH15-transformed tobacco plants. Northern blot analysis showed that the most of the transgenic lines expressed the bgh mRNA. Western blot analysis revealed that the pGAbGH1-transformed tobaccos produced recombinant bGH, but pGAbGH15-transformed ones did not produce the protein. Interestingly, some morphological changes were observed in the roots of transgenic tobacco plants. The transgenic tobacco plants had thick and short roots containing few root hairs in contrast to the non-transformed wild type plants.     

牛脑成纤维细胞生长因子的分离纯化与鉴定

新鲜牛脑组织匀浆液经两步硫酸铵沉淀、CM-Sephadex C50 离子交换层析以及肝素-Sepharose 亲和层析,可得到纯化的酸性和碱性成纤维细胞生长因子(aFGF 和bFGF),分子量分别为13.2kD 和15.2—15.8kD.两种因子均可有效促进3T3细胞的 DNA 合成,ED₅₀分别为15.8ng/ml 和 0.32ng/ml.进一步对 aFGF 的等电点及氨基酸组成做了分析.     

The Study of the Metal-Binding Region in Human Growth Hormone Using Immobilized Metal Ion Affinity Gel-Electrophoresis

The zinc(II)-binding affinities of recombinant human growth hormone and two its mutants, 14–33 and 14–95, were studied using Immobilized Metal Ion Affinity Gel-electrophoresis (IMAG). The mutant hormones, composed of polypeptide chain segments of the human and porcine growth hormones, lacked His18, which may be crucial for binding of the intact hormone to the transition metal ions. The mutations did not affect the affinity of human growth hormone to immobilized zinc ions; the structural analysis implied that the human growth hormone contains two IDA–Zn(II) potential sorption sites formed by amino acid residues His21, Asp171, and Glu174 and/or His18 and Glu174.     

分泌型重组人生长激素工程菌发酵及其产物纯化

在 5L发酵罐中将工程菌pET-ompA3-hGH/BL21(DE3)进行补料式发酵 ,诱导表达分泌型重组人生长激素。通过对发酵培养基成分、补料成分、补料体积、补料时机和滴加流速的选择 ,分泌表达rhGH的量可达到 250mg/L ,占周质蛋白的 44%～ 54% ,发酵周期缩短为8h。用rhGH纯蛋白免疫动物制备的多克隆抗体制成的抗体亲和层析柱 ,一步纯化大肠杆菌周质中分泌表达的rhGH ,产物纯度达到96%以上 ,纯化产物在分子质量大小、免疫活性和二硫键的形成与天然生长激素相一致 。     

牛促卵泡激素在毕赤酵母中的表达

目的:实现重组牛促卵泡激素在毕赤酵母中的表达。方法:依据毕赤酵母的密码子偏爱性设计并利用PCR方法合成了牛促卵泡激素的α亚基和带有6×his-tag的β亚基相应的DNA序列,构建表达载体pHIL-S-bFSH,转化毕赤酵母,表达蛋白进行ELISA定量和Western blot鉴定。结果:重组牛促卵泡激素的表达量为0.26mg/L,Western blot鉴定分子量为35kDa。结论:在毕赤酵母中实现了具有免疫源活性的重组牛促卵泡激素的表达。     

MspI Allelic Pattern of Bovine Growth Hormone Gene in Indian Zebu Cattle (Bos indicus) Breeds

The MspI allelic variation in intron III of the bovine growth hormone (bGH) gene was explored using PCR-RFLP in 750 animals belonging to 17 well-recognized breeds of Indian zebu cattle (Bos indicus) reared in different geographic locations of the country. Restriction digestion analysis of a 329-bp PCR fragment of the bGH intron III region with MspI restriction enzyme revealed two alleles (MspI− and MspI+) and two genotypes (−/− and +/−) across the 17 cattle breeds studied. The allelic frequency varied from 0.67 to 0.94 for MspI (−) and from 0.06 to 0.33 for MspI (+) across the 17 breeds, with a combined average frequency of 0.87 and 0.13, respectively. No animal with +/+ genotype was detected across the samples analyzed. The chi-square test showed that the difference in MspI allelic frequency was not significant (p > 0.05), regardless of the geographic origin, coat color, or utility of the cattle breed. The high MspI (−) allele frequencies obtained for Indian zebu cattle in this study are in sharp contrast to those reported for taurine breeds from northern Europe, Mediterranean countries, and America. Findings of this study further substantiate the hypothesis that the MspI (−) allele has an Indian origin.     

双抗体免疫沉淀法纯化牛生长激素poly(A)~+RNA的研究

我们用双抗体免疫沉淀法,从牛垂体多聚核糖体中分离出牛生长激素特异多聚核糖体,由此多聚核糖体纯化的牛生长激素Poly(A)~+RNA,可以在麦胚体外翻译系统中和兔网织红细胞体外翻译系统中促进~(14)C-亮氨酸的参入。合成的含~(14)-亮氨酸的翻译产物中有91％可以被牛生长激素抗体沉淀。用SDS-11％PAGE对翻译产物进行鉴定表明,翻译产物在25KD处呈一条放射自显影带,与报导的牛生长激素前体分子量相吻合。     

生长激素分泌促进剂及构效关系研究进展

生长激素分泌促进剂是一类作用于垂体和下丘脑的具有专一性促生长激素释放作用的寡肽及其类似物.由于其分子质量小、活性高、可口服、作用专一而有可能成为新的生长激素治疗药物.目前已经发展了很多具有此类活性的多种结构的化合物,如肽、环肽、肽醇及非肽类似物等.尽管这类化合物的作用机制尚未完全明确,但已有证据表明存在新的调节生长激素分泌的途径和新的调节因子.     

青鱼生长激素cNDA的克隆与序列分析

从青鱼（Mylopharyngodon piceus）脑下垂体中提取总RNA,采用逆转录PCR法扩增出青鱼生长激素cDNA编码区,克隆到Pucm-T载体上。序列测定表明青鱼生长激素基因的开放阅读框架含有630bp,其中包括22个氨基酸的信号肽和188个氨基酸的成熟肽,青鱼生长激素基因的克隆成功为该基因的功能及应用研究奠定了基础。