Cloning and Sequence Analysis on cDNA of Growth Hormone Releasing Hormone Receptor Gene from Wuzhishan Miniature Pig

[Objective] cDNA of growth hormone releasing hormone （GHRH） receptor gene from Wuzhishan miniature pig was cloned and its sequence was also analyzed. [Method] Using genomic DNA extracted from porcine ear tissues of Wuzhishan miniature pig as the template, three pairs of primers were designed by the reported cDNA sequence of porcine GHRH, and cDNA was also amplified by RT-PCR. After being recovered and purified, PCR products were ligated to pMD18-T and then transformed into Escherichia coli DH5a. The transformation products were analyzed by PCR and double enzyme digestion to screen positive clones, and the positive clones were sequenced after identification in LB liquid medium. [ Result] cDNA of Wuzhishan miniature pig GHRH receptor gene was obtained successfully, and its length was 1 577 bp coding 423 amino acids. BLAST analysis showed that there were only 23 nuoleotides in difference between this fragment and pomine GHRH receptor gene, and its homology was 98%. However, both GHRH receptor genes were constituted by 423 amino acids with the sequence homology of 96%. [ Conclusion] This study provides theoretical basis for further studies on the dwarf mechanism of Wuzhishan miniature pig.

Gastric motor effects of ghrelin and growth hormone releasing peptide 6 in diabetic mice with gastroparesis

AIM:To investigate the potential therapeutic significance of ghrelin and growth hormone releasing peptide 6 (GHRP-6) in diabetic mice with gastric motility disorders. METHODS: A diabetic mouse model was established by intraperitoneal (ip) injection of alloxan. Diabetic mice were injected ip with ghrelin or GHRP-6 (20-200 μg/kg), and the effects on gastric emptying were measured after intragastric application of phenol red. The effect of atropine, NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) or D-Lys3-GHRP-6 (a growth hormone secretagogue receptor (GHS-R) antagonist) on the gastroprokinetic effect of ghrelin or GHRP-6 (100 μg/kg) was also investigated. The effects of ghrelin or GHRP-6 (0.01-10 μmol/L) on spontaneous or carbachol-induced contractile amplitude were also investigated in vitro, in gastric fundic circular strips taken from diabetic mice. The presence of growth hormone secretagogue receptor 1a transcripts in the fundic strips of diabetic mice was detected by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: We established a diabetic mouse model with delayed gastric emptying. Ghrelin and GHRP-6 accelerated gastric emptying in diabetic mice with gastroparesis. In the presence of atropine or L-NAME, which delayed gastric emptying, ghrelin and GHRP-6 (100 μg/kg) failed to accelerate gastric emptying. D-Lys3-GHRP-6 also delayed gastric emptying induced by the GHS-R agonist. Ghrelin and GHRP-6 increased the carbachol-induced contractile amplitude in gastric fundicstrips taken from diabetic mice. RT-PCR confirmed the presence of GHS-R mRNA in the strip preparations. CONCLUSION: Ghrelin and GHRP-6 increase gastric emptying in diabetic mice with gastroparesis, perhaps by activating peripheral cholinergic pathways in the enteric nervous system.

Isolation of Growth Hormone-Releasing Hormone and Its Receptor Genes from Scatophagus argus and Their Expression Analyses

The teleost Scatophagus argus is a species whose females grows faster than males.Growth hormone(gh)mRNA abundance in females pituitary is higher than that in males;however the mechanism underlining such differential is still unknown.Growth hormone(GH)is tightly associated with GH-releasing hormone(Ghrh)in vertebrates.In this study,Ghrh gene(ghrh)and its receptor gene,ghrhr,were isolated from S.argus.Tissue expression analysis showed that ghrh and ghrhr were mainly expressed in hypothalamus while ghrhr was expressed in pituitary and gh was predominantly expressed in pituitary.Twenty cultured S.argus individuals were used to compare ghrh,ghrhr and gh mRNA abundances,120 g and 181 g average weight for male(n=11)and female(n=9),respectively.Real-time PCR indicated that the ghrh and ghrhr mRNA abundances in male hypothalamus were significantly higher than those in female hypothalamus while that of gh mRNA abundance was significantly higher in female pituitary than in male pituitary.The ghrh and ghrhr mRNA abundances were significantly up-regulated in female hypothalamus 3 h after injection of 0.1 mg kg^-1 body weight Ghrh while pituitary ghrhr and gh mRNA abundances were not affected.In female hypothalamus,ghrh and ghrhr m RNA abundances were not affected at 6 h post-injection of 4 mg kg^-1 body weight 17α-methyltes-tosterone(17α-MT)or 17β-Estradiol(E2).In female pituitary,ghrhr m RNA abundance was down-regulated by 17α-MT while that of gh m RNA abundance was up-regulated by E2.Our findings indicated that E2,rather than Ghrh,plays an important role in up-regulating the expression of gh in female S.argus,which should aid to understand the sexual dimorphism of teleost growth.

Effect of growth hormone-releasing peptide on cardiac cholinergic nerve fiber density distribution in a rat model of heart failure

BACKGROUND： Changes in the cardiac autonomic nerve are considered to be important factors in the mechanisms of heart failure. It is possible to reduce or slow down nerve degeneration and necrosis, provided that patients take effective neuroprotectants during the early stages of heart failure. Moreover, it is possible to relieve the pathological process and reduce the risk of death. OBJECTIVE： To study the effect of growth hormone releasing peptide （GHRP） on cardiac cholinergic nerve fiber density distribution in a rat model of heart failure, and verify whether GHRP can ameliorate denervation. DESIGN, TIME AND SETTING： A randomized controlled study was performed at the Key Laboratory of Anatomy, Harbin Medical University, between June and October 2009. MATERIALS： Fifty adult, healthy, female, Wistar rats, weighing （200± 20） g, were randomly divided into GHRP （n = 30）, model （n = 10）, and sham operation （n = 10） groups. GHRP-2 was made in Shanghai, China （batch No. z071212-03）. METHODS： Acute myocardial infarction was established by ligating the left anterior descending coronary artery in the GHRP and model groups. Five weeks later, myocardial function was detected using color ultrasound electrocardiograph a successful marker of chronic heart failure models Ejection fraction 〈 60% was considered to be However, the left anterior descending coronary artery was not ligated in the sham operation group. The GHRP group was injected with 100 μ g/kg GHRP-2, and the other two groups were injected with the same volume of physiological saline, once per day. MAIN OUTCOME MEASURES： After 4 weeks, pathological changes in cardiac cholinergic nerve fibers were detected under optic microscopy following hematoxylin/eosin staining. In addition, density distribution was measured using a multi-function color pathological image system. RESULTS： In the sham operation group, myocardial cells were regular, uniformly stained, and no inflammatory cells were present. In the model group, myocardial cells were unevenly stained, exhibited nuclear atrophy, degeneration, dissolution, or disappearance. In the GHRP group, myocardial damage was less than in the model group; cardiac muscle fibers exhibited slight degeneration. The myocardium in the sham operation group was serried, spreading the cholinergic innervations along the cardiac fiber. In the model group, there was a decreased number of cholinergic nerve fibers decreased, which also became shorter and smaller, compared with the sham operation group （P 〈 0.01）. In the GHRP group, cholinergic positive nerve fibers were significantly increased compared with the model group （P 〈 0.01）, but still less than the sham surgery group （P 〈 0.05）. CONCLUSION： GHRP delayed denervation and reduced nerve reconstitution following heart failure in rats.

The Role of Protein Kinase C and Its Effect on GHRH in the Regulation of Hormone Secretion by Somatotrophinomas

Summary: Phorbol ester-induced release of growth hormone (GH) and prolactin (PRL) from hu- man somatotrophic tumors was examined in vitro. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) strongly stimulated GH and PRL secretion and showed an additive effect on GH secretion if used in combination with GH releasing hormone (GHRH). In contrast, staurosporine exerted a variable inhibitory effect on GH release. There was no correlation between such effects and gsp mutations. The findings suggested that TPA doesn't act directly through cAMP signal transduction system.

Preclinical therapy of benign prostatic hyperplasia with neuropeptide hormone antagonists

Benign prostatic hyperplasia(BPH)is a pathologic condition of the prostate described as a substantial increase in its number of epithelial and stromal cells.BPH may significantly reduce the quality of life due to the initiation of bladder outlet obstruction and lower urinary tract syndromes.Current medical therapies mostly consist of inhibitors of 5α-reductase orα1-adrenergic blockers;their efficacy is often insufficient.Antagonistic analogs of neuropeptide hormones are novel candidates for the management of BPH.At first,antagonists of luteinizing hormone-releasing hormone(LHRH)have been introduced to the therapy aimed to reduce serum testosterone levels.However,they have also been found to produce an inhibitory activity on local LHRH receptors in the prostate as well as impotence and other related side effects.Since then,several preclinical and clinical studies reported the favorable effects of LHRH antagonists in BPH.In contrast,antagonists of growth hormone-releasing hormone(GHRH)and gastrin-releasing peptide(GRP)have been tested only in preclinical settings and produce significant reduction in prostate size in experimental models of BPH.They act at least in part,by blocking the action of respective ligands produced locally on prostates through their respective receptors in the prostate,and by inhibition of autocrine insulin-like growth factors-Ⅰ/Ⅱand epidermal growth factor production.GHRH and LHRH antagonists were also tested in combination resulting in a cumulative effect that was greater than that of each alone.This article will review the numerous studies that demonstrate the beneficial effects of antagonistic analogs of LHRH,GHRH and GRP in BPH,as well as suggesting a potential role for somatostatin analogs in experimental therapies.

TPA Enhances Growth Horm one (GH) Secretion Effect ofGH-Releasing Horm one (GHRH) by Hum an gsp-PositivePi-tuitary Som atotrophinom as

In recent years, one of the most exciting advances in the researches of pituitary adenomas is the discovery that 30 %-40 % of human pituitary somatotrophinomas carry somatic mutations of the gene for the α subunit of the stimulatory GTP binding protein, G s (G sα). These mutations, termed gsp oncogenes, may play an important role in the tumorigenesis of pituitary adenomas. Of 10 somatotrophinomas examined, 3 (30 %) were proved to be gsp positive, as determined by sequence analysis of DNA generated by the polymerase chain reaction (PCR). GHRH exerted a significant stimulatory effect on GH secretion in 2 of 3 gsp positive and 4 of 7 gsp negative tumors. Moreover, phorbol ester, 1, 2 tetradecanoylphorbol 13 acetate (TPA), enhanced stimulation of lated the GH secretion effect exerted by GHRH in gsp positive somatotrophinomas, whereas this effect was not observed in gsp negative tumors. This result suggests that the protein kinase C signal system as well as adenylyl cyclase cAMP protein kinase A intracellular signal transduction system plays a pivotal role in GH secretory control of GHRH, which may work together via a cross talk mechanism.

Therapeutic effects of ghrelin and growth hormone releasing peptide 6 on gastroparesis in streptozotocin-induced diabetic guinea pigs in vivo and in vitro

Background Diabetic gastroparesis is a disabling condition with no consistently effective treatment. In normal animals, both ghrelin and its synthetic peptide, growth hormone releasing peptide 6 （GHRP-6）, increase gastric emptying. Thus, we investigated the potential therapeutic significance of ghrelin and GHRP-6 in diabetic guinea pigs with gastric motility disorders. Methods A diabetic guinea pig model was produced by intraperitoneal （i.p.） injection of streptozotocin （STZ, 280 mg/kg）. Diabetic guinea pigs were injected i.p. with ghrelin or GHRP-6 （10-100 μg/kg）, and the effects on gastric emptying were measured after intragastric application of phenol red. The effect of atropine or a growth hormone secretagogue receptor （GHS-R） antagonist, D-Lys^3-GHRP-6, on the gastroprokinetic effects of ghrelin or GHRP-6 （100 μg/kg） was also investigated. Further, the in vitro effects of ghrelin or GHRP-6 （0.01-10 μmol/L） on spontaneous or carbachol-induced contractile amplitude in gastric fundic circular strips taken from diabetic guinea pigs were examined. Growth hormone secretagogue receptor transcripts in the fundic strips of diabetic guinea pigs were detected by reverse transcriptase polymerase chain reaction （RT-PCR）. Results We established a guinea pig model of delayed gastric emptying. Ghrelin （20, 50, or 100 μg/kg） and GHRP-6 （20, 50, or 100 μg/kg） accelerated gastric emptying in diabetic guinea pigs with gastroparesis （n=6, P 〈0.05）. In the presence of atropine, which delayed gastric emptying, ghrelin and GHRP-6 （100 μg/kg） failed to accelerate gastric emptying （n=6, P 〈0.05）. D-Lys^3-GHRP-6 also delayed gastric emptying induced by the GHS-R agonist （n=6, P 〈0.05）. Ghrelin and GHRP-6 increased the carbachol-induced contractile amplitude in gastric fundic strips taken from diabetic guinea pigs （n=6, P〈0.05）. RT-PCR confirmed the presence of GHS-R mRNA in the strip preparations. Conclusions Ghrelin and GHRP-6 increased gastric emptying in diabetic guinea pigs with gastroparesis, potentially, by activating the peripheral cholinergic pathways in the enteric nervous system.

Effects of gonadotropin-releasing hormone antagonists on the expression of vascular endothelial growth factor and its receptors in a rat model of ovarian hyperstimulation syndrome

Background Ovarian hyperstimulation syndrome （OHSS） is one of the most life-threatening complications of assisted reproduction treatments. Gonadotropin-releasing hormone antagonists （GnRHanta） are thought to be effective in preventing this complication, and some clinical trials have found lower incidences of OHSS in patients treated with GnRHanta. Our aim was to investigate the effects of GnRHanta on vascular permeability and the expression of vascular endothelial growth factor （VEGF） and its receptors in a rat model of OHSS. Methods An immature early OHSS rat model was established. Three ovarian stimulation protocols were used： pregnant mare serum gonadotropin/human chorionic gonadotropin （hCG） alone, with a GnRHanta, or with a gonadotropin-releasing hormone agonists （GnRHa）. Blood and tissue samples were collected at 48 hours after hCG administration. Vascular permeability was evaluated by measuring the Evans-Blue content of extravasated peritoneal fluids. The expression of VEGF and its receptors, including fit-1 and KDR, were detected by reverse transcriptase-polymerase chain reaction and Western blotting. Results Treatment with both a GnRHanta and a GnRHa resulted in significant reductions in serum estradiol and peritoneal vascular permeability, as well as decreased ovarian expression of VEGF and its two receptors. However, GnRHanta treatment caused a greater reduction in serum estradiol concentrations, and in VEGF receptor mRNA expression than GnRHa. There were no significant reductions in the expression of VEGF or its receptors in extra-ovarian tissues, including the liver, lungs and peritoneum. Conclusion Our results reveal that GnRHanta are more potent than GnRHa in preventing early OHSS through down-regulation of the expression of VEGF and its receptors in hyperstimulated ovaries.

USE OF HOEC ACTIVE ESTERS IN SOLIDPHASE PEPTIDE SYNTHESIS——THE SYNTHESIS OF GROWTH HORMONE RELEASING PEPTIDE

N-hydroxysuccinimide (HOSU) active esters have gained wide application in peptide synthesis, especially in the synthesis of longer peptides by segment conden sation, for they can avoid racemization during coupling processes. However, the HOSU active ester method is prone to side reactions, forming undesirable by-products such as succinimide-oxycarbonyl-alanine N-hydroxysuccinimide ester (Ⅰ) in activating steps and compound (Ⅱ) in the coupling steps sterically hindered by amino acids (proline, valine, isoleucine, etc.).

Role of Gonadotropin-releasing Hormone Stimulation Test in Diagnosing Gonadotropin Deficiency in Both Males and Females with Delayed Puberty

Background： Delayed puberty can result either from constitutional delay of growth and puberty （CDP） or idiopathic hypogonadotropic hypogonadism （IHH）. Gonadotropin-releasing hormone （GnRH） stimulation test has been generally accepted as a current method for diagnosing delayed puberty. The objective of this research was to assess the cut-offvalues and the efficacy of GnRH stimulation test in the diagnosis of delayed puberty in both males and females. Methods： A study of 91 IHH, 27 CDP patients, 6 prepubertal children, and 20 pubertal adults was undertaken. Blood samples were obtained at 0, 30, 60, and 120 rain after GnRH administration and the levels of luteinizing hormone （LH） and follicle-stimulating hormone （FSH） were rneast, red. For each paralneter, the sensitivities and specificities were estimated, and the receiver operating characteristic （ROC） curves were constructed. Resulis： The ROC curves indicated that a serunl basal LH 〈0.6 IU/L or peak LH 〈9.74 IU/L resulted in moderate sensitivity （73.8% or 80.0%） and specificity （90.9% or 86.4%） in the diagnosis of HH in males. Serum basal LH 〈0.85 IU/L or basal FSH 〈2.43 IU/L resulted in moderate sensitivity （80.0% or 100.0%） and specificity （75.0% or 50.0%） in the diagnosis of HH in females. Conclusions： Our data suggest that isolated use of the gonadorelin stimulation test is almost sufficient to discriminate between HH and CDP in males, but unnecessary in females. The most useful predictor is serum basal or peak LH to differentiate these two disorders in males, but serum basal LH or FSH in females.

山羊GHRH-R基因SNPs的快速筛查及基因频率估算

为进一步开展山羊GHRH-R基因的表达调控、分子标记辅助育种及比较基因组学等的研究,采用构建DNA池并用直接测序的方法,研究了黔北麻羊、内蒙古绒山羊和关东奶山羊3个山羊品种GH-RH-R基因的多态性,并估算其等位基因频率。结果表明:在黔北麻羊GHRH-R基因中发现2个SNPs(T-640-C,G-811-T),第8内含子640bp、811bp处分别发生了T→C、C→T碱基转换;等位基因频率T-640为0.500,C-640为0.500,C-811为0.387,T-811为0.613;关中奶山羊和内蒙古白绒山羊在640bp、811bp处没有发生碱基突变。

大口黑鲈GHRH基因启动子区域序列分析及其活性检测

生长激素释放激素(growth hormone releasing hormone,GHRH)是下丘脑弓状核合成和分泌的小分子多肽,其主要功能是调节垂体细胞合成和释放生长激素。为研究大口黑鲈(Micropterus salmoides)GHRH基因5’侧翼启动子区域的活性和该区域中潜在的转录因子对GHRH基因表达的调控作用,对该基因5’端启动子区域约1400 bp长度的片段进行序列分析,预测顺式作用元件,获得了Oct-1、SP1、NF-1、C/EBPalp和C/EBP等多个潜在的调控GHRH基因表达的调节因子结合位点序列。在包括外显子1和内含子1的GHRH基因5’侧翼区两侧加入两个限制性酶切位点Xho I和Bam H I,对其进行改造,并将该片段插入红色荧光蛋白报告基因载体p DsRed2-1,构建了重组表达质粒pGHRH1-RFP。同时,用不含有外显子1和内含子1的GHRH基因5’侧翼区构建重组表达质粒p GHRH-RFP。将质粒pGHRH1-RFP和p GHRH-RFP转染鲤(Cyprinus carpio)上皮细胞(epithelioma papillosum cyprinid,EPC)。经过48 h的培养,在pGHRH1-RFP转染的部分细胞中检测到红色荧光蛋白表达。又将pGHRH1-RFP或p GHRH-RFP质粒注射到斑马鱼(Danio rerio)一细胞或二细胞期的胚胎中,注射了pGHRH1-RFP的胚胎在受精后48 h约有22.5%能检测到有红色荧光蛋白表达,受精后72 h约有29%的仔鱼检测到红色荧光蛋白表达。实验结果表明,目前分离到的GHRH基因5’侧翼序列具有启动基因表达的活性,且该基因的内含子1和外显子1是启动子的活性所必需的。另外,pGHRH1-RFP质粒注射的斑马鱼胚胎只能在胚胎和仔鱼的脊椎和肌肉中检测到RFP的表达,而在脑中没有检测到表达。推测扩增到的大口黑鲈GHRH启动子序列1407 bp(-1043 bp^362 bp)只是起到了驱动RFP脊椎和骨骼肌表达的作用,而不包括驱动在脑组织中特异性表达的启动子,本研究为GHRH基因功能的深入分析奠定了基础。

猪血清中生长激素(GH)和生长激素释放激素(GHRH)的发育性变化研究

采用ELISA方法检测了3个不同体重梯度(90、110和130kg)的川藏黑猪配套系和DLY猪血清中生长激素(GH)和生长激素释放激素(GHRH)的含量变化,并分析了其与肌纤维面积(CSA)和肌内脂肪(IMF)含量的关系。结果显示,90、110和130kg体重梯度川藏黑猪配套系和DLY猪血清中GH含量分别为14.25、14.61、14.11及17.86、16.98、16.77μg/L,GHRH含量分别为22.88、23.98、24.33及27.72、27.47、28.39μg/L,除DLY猪90和130kg血清中GH含量差异显著(P<0.05)外,品种内不同体重梯度间差异均不显著(P>0.05);不同猪种间,除110kg梯度GHRH水平差异不显著(P>0.05)外,其余川藏黑猪配套系均显著低于DLY猪(P<0.05)。相关性分析结果显示,GH、GHRH水平与CSA呈显著正相关(P<0.05),与IMF含量呈极显著负相关(P<0.01)。结果表明GH和GHRH可能通过血液对猪肌纤维的生长和肌内脂肪的沉积进行调控。

肌肉异位表达pGHRH的两种质粒对大鼠的促生长作用

构建了两种猪生长激素释放激素(GHRH)的肌肉特异表达质粒ppG-A53f-H6-GHRH(ppG-H6)及ppG-A53f-H4-GHRH(ppG-H4).通过对大鼠进行肌肉注射,并于注射后分别记录0,3,7,11,14,17,20,24,27,31,34,38以及第41天的体重变化结果,同时分析了第20、27、34和第41天的血清生长激素(GH)浓度,以研究促生长表达质粒在动物体内的促生长作用.通过研究表明,外源的GHRH表达质粒在肌肉中获得了表达,并使动物体内的GH水平维持在一个相对稳定的较高水平上,导致的直观效果表现为:通过肌肉注射表达质粒ppG-H6对大鼠的生长有促进效果,在第34天的净增重显著于对照组((200.57±3.99)gvs(185.85±9.45)g,P<0.05).实验结果预示,利用注射肌肉特异异位表达GHRH蛋白可能促进动物生长.

鸭发育早期下丘脑GHRH、SS mRNA的表达

为比较研究不同鸭种胚胎期和出雏早期下丘脑生长激素释放激素(GHRH)和生长抑素(SS)的表达规律及其与体质量和肝质量的相关性,采用qRT-PCR方法研究了鸭13、17、21、25、27胚龄和出雏后7日龄下丘脑GHRH与SS mRNA的表达变化。结果表明,鸭下丘脑GHRH与SS在13胚龄已有表达;SS在鸭发育早期维持低表达状态,GHRH呈现极显著的品种和时间特异性差异;品种和日龄的交互作用极显著影响早期发育过程中GHRH的表达。体质量和肝质量的变化也呈现极显著的品种和时间特异性差异,但与GHRH表达量的相关性不明显。

GHRHR-SV1抗体的制备及其蛋白在黑色素瘤细胞中的表达

目的:制备生长激素释放激素受体剪接变异体1(GHRHR-SV1)抗体,并检测GHRHR-SV1蛋白在黑色素瘤细胞中的表达情况,为后续的肿瘤机制研究奠定基础。方法:设计GHRHR-SV1基因序列,将其插入质粒pET-32a(+)中,构建pET-GHRHR-SV1重组载体,经PCR及测序鉴定后,转入大肠杆菌BL21(DE3)中,分别加入0、0.5、1.0、1.5和2.0mmol·L^(-1)异丙基-β-D-硫代半乳糖苷(IPTG);诱导1、2、4和6h,并在37℃、30℃和25℃不同温度下进行诱导;观察目的蛋白表达的最佳IPTG浓度、时间和温度。亲和层析纯化重组蛋白,将纯化后的蛋白免疫家兔,制备兔多克隆抗体,ELISA法检测该抗体效价,Western blotting法检测GHRHR-SV1蛋白在黑色素瘤细胞系B16-F10中的表达情况。结果:重组载体pET-GHRHR-SV1构建成功。在IPTG浓度为2.0mmol·L^(-1)时目的蛋白的表达量最高;IPTG诱导蛋白表达2h时,蛋白表达量最大;当温度为25℃时,蛋白的表达量最大。诱导表达的蛋白相对分子质量约为24 000,纯化后纯度为80%,GHRHR-SV1抗体效价为1∶1 600 000,B16-F10细胞中有GHRHR-SV1蛋白表达。结论:成功制备pETGHRHR-SV1重组载体。GHRHR-SV1蛋白在IPTG浓度为2.0mmol·L^(-1)、诱导时间为2h和温度为25℃的条件下表达最佳。成功制备了高效价的GHRHR-SV1抗体。黑色素瘤细胞系中有GHRHR-SV1蛋白表达。

gsp癌基因在人垂体生长激素腺瘤的表达及与GHRH对生长激素分泌效应比较

采用聚合酶链反应和直接序列分析法评价了30例肢端肥大症肿瘤组织的gsp癌基因,发现9例30％为gsp癌基因阳性。结果与生长激素释放激素对生长激素分泌效应比较,发现gsp癌基因阳性和阴性肿瘤体外生化特征各自差异较大,推测可能与细胞间多信号传导系统介入有关。

重组生长激素释放激素GHRH的纯化及活性测定

目的用基因工程的方法在大肠杆菌中诱导表达重组生长激素释放激素(GHRH),检测其生物学活性。方法将重组表达质粒pET-28a /L-ansB-GHRH,转化大肠杆菌BL21(DE3),IPTG诱导融合蛋白表达。经裂解细胞、洗涤、乙醇沉淀、酸水解、SP-Sephadex C-25和Sephadex G25柱层析等方法纯化GHRH。用人生长激素放免试剂盒检测GHRH多肽的活性。结果SDS-PAGE分析显示重组表达质粒在大肠杆菌中成功地表达了融合蛋白,它以不溶性的包涵体形式存在,占菌体总蛋白的30%左右。经抽提、酸水解、层析等方法表明纯化倍数达147倍,多肽收率为0.68%。通过电喷雾质谱测得多肽的相对分子质量为5235.25,与理论计算值相吻合。多肽的纯度经SDS-PAGE测定为单一峰。GHRH三组刺激实验组与空白对照组相比之间差异显著(P<0.01),且随着剂量增加,差异明显增加。结论体外重组GHRH多肽具有较高的生物学活性。