重组猪生长激素表达研究进展

猪生长激素作为猪生长发育过程中起主导作用的蛋白质激素,能增加肌肉组织蛋白含量、降低脂肪含量、促进骨骼发育、刺激乳汁分泌等,是畜牧养殖业中值得开发利用的激素之一。利用基因工程技术生产重组猪生长激素使猪生长激素在生产上应用成为可能。本文简要介绍了猪生长激素,总结了重组猪生长激素表达的研究进展,并展望了重组猪生长激素的应用与发展前景。     

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。     

猪生长激素研究进展

猪生长激素是由猪脑垂体前叶嗜酸性细胞分泌的一种单一肽链的蛋白质激素。本文就猪生长激素基因的结构特点,多肽性研究、基因表达及猪生长激素的结构特点、生理功能、分泌规律及与其抗体的相互作用等方面进行了综述。     

在大肠杆菌中温度诱导高效表达猪生长激素

本实验构建了温度诱导的表达猪生长激素的表达载体,转化大肠杆菌N4830后,经温度诱导,获得了高效表达．由SDS-聚丙烯酰胺凝胶电泳及Westerm blot证实表达产物为猪生长激素,经扫描估测猪生长激素的表达量占大肠杆菌细胞全蛋白的30％左右。     

猪生长激素cDNA的全序列分析

本文报道了两个猪生长激素cDNA的全序列分析的结果。猪生长激素cDNA是我们由猪垂体mRNA经过反向转录、克隆和筛选后获得的。文中还将这两个序列与Seeburg等(1983)报道的序列以及Vize等(1987)报道的猪基因组生长激素基因的序列进行了比较和讨论。     

草鱼生长激素单抗免疫亲和柱的制备及初步应用

用纯化的草鱼生长激素单克隆抗体偶联到CNBr活化的Sepharose4B凝胶上,制成约10ml的亲和层析柱．用该柱一步纯化了重组鲤鱼生长激素基因的表达产物．偶联有13．65mg单克隆抗体的亲和柱一次可纯化得到约0．7mg重组鲤鱼生长激素．酶联免疫受体测定表明它具有强烈的生物学活性,SDS－PAGE表明它为单一蛋白带,分子量约为22000．     

草鱼生长激素单抗免疫亲和柱的制备及初步应用

用纯化的草鱼生长激素单克隆抗体联剂ＣＮＢｒ活化的Ｓｅｐｈａｒｏｓｅ ４Ｂ凝胶上制成约１０ｍｌ的亲和层析柱,用该柱一步纯化了重组鲤鱼生长激素基因的表达产物。偶联有１３．６５ｍｇ单克隆抗体的亲和柱一次强纯化得到约０．７ｍｇ重组鲤鱼生长激素。酶联免疫受体测定表明它具有强烈的生物学活性,ＳＤＳ－ＰＡＧＥ表明它为一蛋白带,分子量约为２２０００。     

榕江香猪生长激素基因的鉴定及功能分析

生长激素是调节动物生长的主要激素.本研究应用聚合酶链式反应技术从榕江香猪的基因组文库中分离出1.903kb生长激素基因.克隆的生长激素基因由五个外显子和四个内含子组成.榕江香猪生长激素基因的碱基序列与已知四个国外猪种和9个中国地方猪种之间的同源性为97%～99%,其间的差异主要集中在内含子2和4.通过限制性内切酶（DdeI,NarI,BsmNI）分析,鉴定出榕江香猪生长激素基因的五个多态性位点,分别位于5＇-侧翼区274（T/C）位点,外显子2的622（G/A）和631（G/A）位点,内含子2中的841（T/C）以及外显子4中的1 358（A/G）位点.同时,1 358（A/G）位的碱基改变导致榕江香猪生长激素成熟肽第108位异亮氨酸替换,三维结构分析表明,异亮氨酸的存在可能导致生长激素与受体间亲合力降低.     

鲤鱼（Cyprinus carpio）血清生长激素变化的酶联免疫吸附测定

本研究的目标是纯化鲤（Cyprinus carpio）生长激素,用于制备抗鲤鱼生长激素的单克隆抗体,建立鲤鱼生长激素的酶联免疫吸附测定（enzyme-linked immunosorbent assays,ELISA）技术,并用所建立的技术检测不同环境下养殖的鲤鱼的血清生长激素水平的变化．利用柱纯化技术纯化酵母表达草（Ctenopharyngodon idella）生长激素,免疫新西兰白兔（Oryctolagus cuniculus）,获得抗草鱼生长激素多克隆抗体．摘取鲤鱼垂体,从中提取生长激素,经层析纯化后,以变性聚丙烯凝胶电泳判断其分子量和纯度,并利用抗草鱼生长激素多克隆抗体以Western blot验证．纯化并经鉴定的鲤鱼生长激素作为免疫原被用于B淋巴细胞杂交瘤技术．共建立14株能够稳定分泌抗鲤鱼生长激素单克隆抗体的杂交瘤细胞系（FMU-cGH1-FMU-cGH14）,其中8个克隆（FMU．cGH1～FMU-cGH6,FMU-cGH12和FMU-cGH13）成功用于Western blot分析,9个克隆（FMU-cGHl-FMU-cGH7,FMU-cGH9和FMU-cGH10）可用于荧光标记和免疫组织化学分析．利用竞争性ELISA进行表位分析,结果表明这些单克隆抗体能够识别5个不同的表位．利用其中的FMU-cGH12作为包被抗体,FMU-cGH6作酶标抗体,建立了能够检测鲤鱼生长激素含量的ELISA技术．这一检测系统被证明具有高度的稳定性和灵敏度,能够检测并定量低至70pg／mL的生长激素．利用这一检测技术,发现限制进食和网箱养殖的鲤鱼其生长激素含量都有明显提高,分别为对照的6．9和5．8倍,显示不同生长环境下鲤鱼生长激素水平具有不同的反应．     

国际矿产化学公司打算使重组体猪生长激素商品化

国际矿产化学公司(IMC)希望最早在1988年能大规模生产重组体猪生长激素。猪生长激素将是为制造动物产品而建的100万美元的生物技术新设施的第一批产品。如果走运的话,设施的设计和建造将同时进行,届时 IMC 也将完成其开发工作和得到联邦政府的产品许可证。IMC 将根据与 Biogen 公司的许可协议生产生长激素,Biogen 公司几年前为 IMC 克隆了猪和牛的生长激素。     

Immunological relatedness of human and porcine growth hormones.

The immunological relatedness of human and porcine growth hormones is examined by means of labelled human growth hormone and guinea pig antiserum. 1) Labelled human growth hormone is found in the precipitate after reaction with antiserum against porcine growth hormone. Parallel dilution curves are obtained with antisera against human and porcine growth hormones. 2) After addition of antiserum against porcine growth hormone, all the radioactivity is eluted from Sephadex G-100 with the void volume. 3) The addition of an excess of porcine hormone displaces labelled human growth hormone from antibodies against human growth hormone to the same extent as an excess of non-labelled human growth hormone does. 4) The standard radioimmunoprecipitation curves for porcine and human growth hormones obtained in the assay system for the human hormone are parallel in slope, provided that the human hormone and our preparation of the porcine hormone are introduced at a proportion of 1 to 560. 5) In a double diffusion test in agarose gel layers, with human and porcine growth hormones diffusing against guinea pig anti-porcine serum, cross reaction is observed. The conclusion is drawn that with guinea pig antisera, human and porcine growth hormones behave immunologically in a similar fashion. Labelled human growth hormone seems to have only such immunodeterminants as are also found in porcine growth hormone.     

Immunological identity of human and porcine growth hormones. Application to determination of growth hormone in porcine serum.

In two radioimmunoassay systems with iodinated human growth hormone as tracer and anti-human growth hormone or anti-porcine growth hormone as binding site, the standard curves for human and for porcine growth hormone were parallel. In both systems the porcine growth hormone preparation had to be added in about the same excess as compared to the human hormone. It was concluded that the human and the porcine hormones behave in an identical way and that the values obtained in radioimmunoassay for human growth hormone represent the amount of immunologically active molecules in the porcine growth hormone preparation. As parallel standard curves were obtained with porcine serum, the concentration of growth hormone is porcine serum may be determined by radioimmunoassay for human growth hormone.     

Preparation and characteristics of porcine growth hormone.

A recently published method for preparation of porcine growth hormone resulted in a highly purified protein with good biological activity. However, after storing for some months the originally homogeneous hormone separated again into several fractions when rechromatographed on ion exchange columns. The biological activity, found in one of these fractions, was clearly diminished compared with the activity of a freshly prepared hormone. In the present paper a modified procedure is described for the isolation of a more stable porcine growth hormone. The influence of ions, involved in buffers of the same molarity and the same pH, upon ion exchange chromatography of porcine growth hormone is discussed. The purified hormone shows high biological activity in the tibia test and is free of activities of other pituitary hormones. The molecular weight is about 20 000; only phenylalanine is found as N-terminal as well as C-terminal amino acid; the amino acid composition resembles neither that of porcine growth hormone described in literature nor that of human growth hormone.     

Possible evidence for the existence of a growth hormone fragment in porcine pituitary

In an attempt to search for growth hormone fragments in the pituitary, a radioimmunoassay was developed for a 55 residue S-amino-ethylated CNBr fragment (fragment B) of porcine growth hormone corresponding to residues 126–180 of human growth hormone. The assay was sensitive to 50 pg of fragment B whereas displacement of ¹²⁵I-labelled fragment B porcine growth hormone required a 10³ M excess and was non-parallel. In a homogolous porcine growth hormone radioimmunoassay, fragment B was non-reactive. Gel filtration of an extract of porcine pituitary on Sephadex G-75 revealed three peaks of fragment B immunoreactivity: peak I (29% of total immunoreactivity) eluted in the void volume, peak II (49%) eluted in the position of growth hormone, and peak III (12%) was more retarded than fragment B. Nearly all of the growth hormone immunoreactivity eluted as a single peak in the position of ¹²⁵I-labeled porcine growth hormone. The dilution curve of peak III but not of peaks I or II was parallel to that of fragment B. The results indicate the existence within porcine pituitary of material cross-reactive with a portion of the growth hormone molecule, possibly representing a growth hormone fragment.     

Isolation and characterization of the porcine hypothalamic growth hormone releasing factor

A 44 amino acid peptide with high intrinsic growth hormone releasing activity was isolated from 2500 porcine hypothalami by means of acid extraction, immunoaffinity chromatography, gel filtration, and 2 steps of reverse phase HPLC. The growth hormone releasing factor was structurally characterized by gas phase sequence analyses of the intact peptide and its carboxyl terminal cyanogen bromide digestion fragment. Reverse phase liquid chromatography of the native peptide and synthetic replicates showed that the molecule possesses an amide rather than a free acid at its carboxyl terminus. The structure of the peptide was established as: Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Arg-Val-Arg-Leu-NH2 using approximately 6 nmol of material.     

Genetic engineering of peptide hormones

The application of different approaches for preparing DNAs coding for peptide hormones was demonstrated. The libraries of human, bovine and porcine pituitaries cDNA were obtained starting from their total mRNAs. Screening of these libraries revealed clones containing human, bovine and porcine growth hormone sequences, cDNAs for bovine ACTH-beta-lipotropin precursor and for bovine and porcine prolactin. The gene of human calcitonin was created by combination of chemical and enzymatic synthesis. This synthetic gene was further cloned in pBR322. The expression of cloned human growth hormone cDNA under control of different Escherichia coli promoters was studied and physico-chemical and biological properties of the growth hormone produced by E. coli were tested.     

Isolation and characterization of growth hormone from blue fox pituitary glands

Growth hormone has been purified to homogeneity from blue fox pituitary glands. It has 191 amino acids with two disulfide bridges and a single tryptophan residue. The somatotropin activity is only 8% when compared with the bovine hormone in the receptor-binding assay. From radioimmunoassay data using baboon antisera to porcine or bovine growth hormone, the fox hormone has 14-17% immunoreactivity of bovine or porcine hormone.