Molecular endocrinology of oocyte growth and maturation in fish

Pituitary gonadotropins (GTHs) are of primary importance in triggering oocyte growth and maturation. However, the actions of GTHs are not direct, but are mediated by the ovarian production of steroidal mediators of oocyte growth (estradiol-17β) and maturation (maturation-inducing hormone, MIH; 17α,20β-dihydroxy-4-pregnen-3-one, 17α,20β-DP in salmonid fishes; 17α,20β,21-trihydroxy-4-pregnen-3-one, 20β-S in sciaenid fishes). It is established that production of estradiol-17β and 17α,20β-DP by salmonid ovarian follicles occurs via the interaction of two cell layers, the thecal and granulosa cell layers (two-cell type model). A distinct shift in the salmonid steroidogenesis from estradiol-17β to 17α,20β-DP occurs in the ovarian follicle layer immediately prior to oocyte maturation. It is possible that this shift is a consequence of dramatic changes in the expression of the genes encoding various steroidogenic enzymes. As an initial step to address this question, we have isolated and characterized the cDNAs encoding a number of ovarian steroidogenic enzymes including the rainbow trout cholesterol side-chain cleavage cytochrome P-450, 3β-hydroxysteroid dehydrogenase (HSD), 17α-hydroxylase/17,20 lyase cytochrome P-450, aromatase cytochrome P-450 cDNAS as well as the pig 20β-HSD cDNA. Estradiol-17β stimulates the hepatic synthesis and secretion of a yolk precursor, vitellogenin. Vitellogenin is then transported to the ovary where it is selectively taken up into the oocyte by a receptor-mediated process involving specific cell-surface receptors. Estradiol-17β was also shown to induce the synthesis of egg membrane proteins in the liver. The maturation-inducing action of 17α,20β-DP and 20β-S is through the binding to the oocyte plasma membrane. This initial MIH-surface interaction is followed by the formation of the major mediator of MIH, maturation-promoting factor (MPF). We have purified MPF from mature oocytes of carp. Carp MPF consists of two components: the homolog of the cdc2⁺ gene product of fission yeast (p34^cdc2) and cyclin B. The cdc2 kinase protein is present in immature oocytes as well as in oocytes induced to mature by 17α,20β-DP treatment, while cyclin B proteins can be detected only in mature oocytes. Addition of bacterially expressed goldfish cyclin B to the extracts of immature goldfish oocytes induced MPF activation. These results suggest that the appearance of cyclin B protein is a crucial step for 17α,20β-DP-induced oocyte maturation in fish.     

cDNA cloning of goldfish Hsp27 and its chaperone activity

A cDNA fragment encoding goldfish Hsp27 was amplified by polymerase chain reaction and its nucleotide sequence was determined. The deduced amino-acid sequence of goldfish Hsp27 showed 58–62% identity to Hsp27 from other vertebrates. Three serine residues reported to be phosphorylated in human Hsp27 were conserved in goldfish Hsp27. The Hsp27 gene was transcribed in goldfish culture cells after temperature shift from 20°C to 40°C, but not from 20°C to 35°C. Recombinant goldfish Hsp27 was examined for its in vitro chaperone activity and compared to those of Hsp30 from the same fish and human recombinant Hsp27. Goldfish Hsp27 and Hsp30 showed a similar activity at 10 μM, which was significantly lower than that of human Hsp27. At 1 and 5 μM concentrations, however, goldfish Hsp27 showed slightly lower activity than goldfish Hsp30, but the activities of both goldfish proteins still remained significantly lower than that of human Hsp27. Meanwhile, goldfish Hsp27 formed oligomer, which was slightly smaller than that of Hsp30. These results suggest that goldfish Hsp27 contributes as a molecular chaperone in association with Hsp30 to compensate for stress resulting from rapid temperature fluctuations. The nucleotide sequence reported in this paper has been registered into the DDBJ/EMBL/GenBank databases with accession number AB239443 for goldfish Hsp27.     

Production of recombinant goldfish gonadotropins by baculovirus in silkworm larvae

Recombinant gonadotropins (GTH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) of goldfish Carassius auratus were produced by baculovirus in silkworm larvae. Hemolymph containing recombinant FSH (rFSH) or LH (rLH) was collected from silkworm larvae, and its biological activity was examined in vivo using male goldfish and female bitterling Rhodeus ocellatus ocellatus. Injection of hemolymph containing rFSH or rLH induced milt production in male goldfish, whereas only rLH induced ovulation in female bitterling. These results suggest, that biologically active goldfish recombinant GTH could be applied for the induction of gonadal development in aquaculture fishes as a substitute of pituitary extract, if a method of large scale production is established.     

鲤疱疹病毒Ⅱ型ORF66基因DNA疫苗载体的构建及其免疫效果

鲤疱疹病毒Ⅱ型(CyHV-2)能够引起鲫大量死亡,严重威胁我国水产养殖业的健康发展,目前,针对CyHV-2尚无有效的商业化疫苗或治疗措施。为构建CyHV-2DNA疫苗,本研究将其衣壳蛋白ORF66编码基因克隆至pVAX1真核表达载体上,构建重组质粒pVAX-ORF66。此外,在BL21(DE3)pLysS中对ORF66蛋白进行了原核表达,表达蛋白经纯化后免疫新西兰白兔制备了ORF66特异性抗体。酶联免疫吸附(ELISA)检测显示,制备抗体效价达1∶20000以上。将pVAX-ORF66质粒转染金鱼脑细胞系(GFB),利用制备的ORF66抗体进行间接免疫荧光(IFA)检测,结果显示,ORF66蛋白可以在细胞中大量表达,且主要定位于细胞质中。将pVAX-ORF66质粒肌肉注射鲫后进行CyHV-2免疫保护实验,结果表明,其相对免疫保护率达55.6%。本研究针对CyHV-2构建了一种制备简单、成本低廉的DNA疫苗,为鲫造血器官坏死病的免疫预防及感染分子机制研究奠定了前期实验基础。     

Identification and characterization of maturation-promoting factor from catfish,Clarias batrachus

Maturation-promoting factor (MPF) extracted from maturing oocytes of catfishes (Clarias batrachus andHeteropneustes fossilis) and carp (Labeo rohita) induces 100% germinal vesicle breakdown (GVBD) when microinjected intoClarias immature unstimulated oocytes. The presence of a similar MPF activity has also been demonstrated in the active fractions collected after superose 12. SDS-PAGE analyses of cytosolic extracts (CE) prepared from immature and mature oocytes revealed the presence of 34- and 46 kDa proteins apart from a few others. Antibody against the PSTAIR sequence of p34^cdc2 recognized 32- and 34 kDa proteins of immature as well as mature oocytes while, 46 kDa protein of mature oocytes was recognized by anti-cyclin B1 antibody. Moreover, labelling of [³⁵S]methionine was observed mainly in the region of 46 kDa protein band indicatingde novo synthesis of this particular protein. Anti-cyclin A antibody did not recognize any proteins of immature or mature oocytes. Cyclin B1 was absent in immature oocytes and ovulated eggs. These findings indicate the presence of p34^cdc2 homologs and cyclin B in the MPF of the catfishes and carp oocytes.     

Analysis of the α-amylase gene sequence and the enzyme activity of Indian rock oyster Saccostrea forskali

Indian rock oyster Saccostrea forskali is an important commercial species in Thailand. In this study, its full-length α-amylase (SfAmy) cDNA nucleotide sequence was investigated. The SfAmy cDNA was 1,689 bp long and contained a 1,563-bp open reading frame encoding 520 amino acid residues, including a 17-amino acid signal peptide. The molecular mass and the estimated isoelectric point (pI) of the deduced mature S. forskali α-amylase (SfAMY) were 55.948 kDa and 6.45, respectively. The deduced protein sequence showed 45–88 % identity to other mollusk AMYs. The molecular weight was confirmed by the weight of the purified native enzyme. The specific activities of crude and purified native enzymes toward 1 % starch were 29.53 and 187.42 U/mg. In addition, the obtained recombinant SfAMY also showed activity in digesting 1 % starch. The specific activities of the crude and purified recombinant proteins were 11.8 and 46 U/mg. Both enzymes showed optimal activity temperature at 40 °C but their optimum pH values were different, 6.0 for the native and 5.0 for the recombinant. The expression of SfAmy examined by RT-PCR showed the highest levels in the digestive gland but none was observed in the adductor muscle.     

雌核发育二倍体鲫鲤及其他倍性鱼cdc2基因cDNA全序列克隆及表达分析

为研究雌核发育二倍体鲫鲤产生二倍体卵子的分子机制,实验采用PCR和cDNA末端快速分离法,克隆获得了雌核发育二倍体鲫鲤第三代(G3)、二倍体红鲫、三倍体湘云鲫和四倍体鲫鲤的细胞周期相关基因——cdc2基因cDNA全序列.结果显示,4种不同倍性鱼cdc2基因均编码含有302个氨基酸蛋白,而且编码的蛋白都含有与其他CDK激酶相当保守的序列PSTAVRE;同源性分析发现,4种鱼cdc2基因编码的氨基酸序列之间的相似度大于97.6％,说明Cdc2蛋白在这4种不同倍性鱼中具有高度保守性.采用实时荧光定量PCR(real-time PCR)对cdc2基因在G3、二倍体红鲫、三倍体湘云鲫及四倍体鲫鲤早期卵巢中的表达进行分析,结果发现,G3cdc2基因比普通二倍体红鲫和三倍体湘云鲫表达要高,比四倍体鲫鲤的表达水平低.该研究从分子水平证明了G3早期性腺中存在着大量的多倍体卵原细胞.同时,研究表明,cdc2基因在雌核发育二倍体鲫鲤早期卵巢的高表达暗示着G3多次进入S期却不经历M期导致二倍体配子的产生.     

The purification and partial characterization of carp,Cyprinus carpio,vitellogenin

A procedure is described for the isolation of intact vitellogenin (c-VTG) from the carp, Cyprinus carpio. VTG was induced in juvenile females using oestradiol-17β and purified from the plasma using a combination of gel-filtration chromatography on Sepharose 6B and ion exchange chromatography on DEAE-cellulose. Purification procedures were conducted at low temperatures (below 9°C) in the presence of the proteolytic enzyme inhibitor aprotinin to prevent degradation. Intact c-VTG had an apparent molecular mass of 390,000 Daltons, but when extracted from plasma in the absence of aprotinin it underwent proteolysis into at least 2 protein fragments (apparent molecular masses of 230,000 and 96,000 Daltons), showing an instability of the native dimer. An amino acid analysis of c-VTG showed that its composition was almost identical to goldfish VTG, a species closely allied to the true carps and also similar to other oviparous vertebrate VTGs. Collectively, these data indicate that using these purification procedures VTG from carp, and probably other teleost species, can be isolated in an intact, highly purified form.     

Cannabinoid receptors are widely expressed in goldfish: molecular cloning of a CB2-like receptor and evaluation of CB1 and CB2 mRNA expression profiles in different organs

Cannabinoids, the bioactive constituents of Cannabis sativa, and endocannabinoids, among which the most important are anandamide and 2-arachidonoylglycerol, control various biological processes by binding to specific G protein-coupled receptors, namely CB1 and CB2 cannabinoid receptors. While a vast amount of information on the mammalian endocannabinoid system does exist, few data have been reported on bony fish. In the goldfish, Carassius auratus, the CB1 receptor has been cloned and its distribution has been analyzed in the retina, brain and gonads, while CB2 had not yet been isolated. In the present paper, we cloned the goldfish CB2 receptor and show that it presents a quite high degree of amino acid identity with zebrafish Danio rerio CB2A and CB2B receptors, while the percentage of identity is lower with the puffer fish Fugu rubripes CB2, as also confirmed by the phylogenetic analysis. The sequence identity becomes much lower when comparing the goldfish and the mammalian CB2 sequences; as for other species, goldfish CB2 and CB1 amino acid sequences share moderate levels of identity. Western-blotting analysis shows the CB2 receptor as two major bands of about 53 and 40 kDa and other faint bands with apparent molecular masses around 70, 57 and 55 kDa. Since the distribution of a receptor could give information on its physiological role, we evaluated and compared CB1 and CB2 mRNA expression in different goldfish organs by means of qReal-Time PCR. Our results show that both CB1 and CB2 receptors are widely expressed in the goldfish, displaying some tissue specificities, thus opening the way for further functional studies on bony fish and other nonmammalian vertebrates.     

鲫卵黄脂磷蛋白的纯化鉴定及其夹心ELISA的建立

鲫(Carassius carassius)卵黄原蛋白(vitellogenin,Vtg)是检测水体环境雌激素活性常用的生物标志物。本研究利用凝胶过滤结合离子交换层析与选择性沉淀结合离子交换层析2种方法,从鲫卵巢匀浆液中纯化得到了卵黄脂磷蛋白(lipovitellin,Lv),经鉴定该蛋白含有糖、磷、脂基团,天然分子量约为521 kD,SDS变性电泳显示分子量为117 kD和103 kD的2个亚基。Western blot结果显示,金鱼(Carassius auratus)Lv抗体和斑马鱼(Danio rerio)Lv抗体都能与鲫Lv发生很好的交叉反应。利用纯化的鲫Lv与金鱼Lv抗体和斑马鱼Lv抗体建立了2种夹心ELISA,发现金鱼Lv和鲫Lv的结合曲线基本重合,并且利用鲫Lv与金鱼Lv抗体建立的夹心ELISA工作范围为15.6~1000 ng/mL,检出限约为6.8 ng/mL,显著低于利用斑马鱼Lv抗体建立的夹心ELISA,结合此前研究者建立的鲫Vtg竞争ELISA,为鲫Vtg指标的测定提供了可靠方法。     

Identification of a protein kinase which phosphorylate α4 subunit of the 26S proteasome in goldfish oocytes

To investigate the regulatory mechanism for the proteasome in the meiotic cell cycle, we purified the 26S proteasome from immature (in G2-phase) and mature (in M-phase) oocytes, and compared its subunits by immunoblotting. A monoclonal antibody, GC3β (anti-goldfish 20S proteasome component 3β) cross-reacted with two bands in the 26S proteasome from immature oocytes, however the upper band was absent in the 26S proteasome from mature oocytes. cDNAs which encode the α4 subunit of goldfish 20S proteasome (α4 _ca ) were isolated by an immuno-screening method using GC3β. Phosphatase treatment of the 26S proteasome revealed that a part of α4 _ca phosphorylated in G2-phase and dephosphorylated in M-phase. By the assay using recombinant α4 _ca as a substrate, a kinase was purified by column chromatographs. Amino acid sequence analysis was performed for resulting partial purified fraction. A protein band, which well corresponded to the kinase activity, was identified as Casein kinase-1α (CK-1α). The result suggests that CK-1α phosphorylate α4 subunit of the 26S proteasome in immature oocyte of goldfish. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of biologically-active recombinant goldfish gonadotropins in transgenic rainbow trout

The feasibility of using rainbow trout Oncorhynchus mykiss embryos as an expression system for proteins was investigated. For model proteins, we selected two goldfish gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH). To produce single-chain goldfish FSH (scgfFSH) and LH (scgfLH), cDNAs encoding glycoprotein hormone (GP) α and FSHβ were fused in tandem, and cDNAs encoding GPα and LHβ were fused in tandem. The fused cDNAs were ligated with β-actin promoter, and microinjected into fertilized rainbow trout eggs. After 4-days incubation, the embryos were subjected to western blotting and in vitro bioassays. The recombinant proteins produced by the embryos were immunoreactive to antisera against goldfish GPα, N-glycosylated, and biologically active. We conclude that scgfFSH and scgfLH were successfully produced in transgenic rainbow trout.     

Hormonal influences on in vitro [35S]-sulfate uptake by gill arches from the goldfish (Carassius auratus L.)

A bioassay for insulin-like growth factor (IGF), based on the in vitro incorporation of [³⁵S]-sulfate into gill arch tissue was used to study the hormonal regulation of proteoglycan synthesis in the goldfish (Carassius auratus). [³⁵S]-sulfate incorporation into gill arch tissue was found to be time-dependent with maximal uptake occurring by 48h, suggesting that proteoglycan synthesis in this tissue was maintained for at least 48h in vitro. The addition of human recombinant IGF-I (IGF-I) to the incubation medium was found to significantly stimulate [³⁵S]-sulfate uptake into the gill arches, whereas bovine growth hormone (GH) was without effect. Porcine insulin was also stimulatory, but results indicate that the effects of porcine insulin and IGF-I may be mediated by a single receptor system. Finally, arches from hypophysectomized fish were significantly less responsive to IGF-I than were arches from sham-operated fish. Furthermore, administration of ovine GH in vivo appeared to increase subsequent responsiveness in vitro. Together, these results provide evidence that the growth-promoting actions of GH in the goldfish may be mediated, at least in part, by a peptide related in structure to mammalian IGF-I.     

急性病毒性坏死病毒IAP-86基因的克隆、表达及抗凋亡研究

在已经完成的栉孔扇贝急性病毒性坏死病毒(acute viral necrosis virus,AVNV)全基因组序列测序与分析的基础上,设计特异性引物,克隆得到了ORF86编码的杆状病毒凋亡抑制蛋白基因(IAP-86)。IAP-86基因与pET32a(+)质粒连接构建得到重组质粒pET32a-IAP86,将重组质粒转化到E.coil BL21(DE3)中,使用异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达,SDS-PAGE检测显示表达蛋白分子量约为40 ku,经Western-blotting和质谱分析证明,该蛋白即为IAP-86融合蛋白,Co2+柱纯化后得到了纯化的IAP-86融合蛋白。将重组的IAP-86蛋白用FITC标记,荧光显微镜下观察,发现重组的IAP-86蛋白最终能够与栉孔扇贝血淋巴细胞的细胞核和细胞质结合。细胞凋亡检测实验发现,重组的IAP-86蛋白能够在一定程度上抑制栉孔扇贝血淋巴细胞凋亡,凋亡抑制率为7%。本实验应用原核表达成功得到了IAP-86蛋白,并证明IAP-86对栉孔扇贝细胞的凋亡有一定抑制作用,这为进一步研究AVNV的侵染机制提供依据。     

中华绒螯蟹精巢特异表达蛋白DMRT-like:抗体制备及免疫鉴定

Dmrt是性别调控因子Doublesex和Mab-3的相关基因,近年报道了中华绒螯蟹EsDmrt-like只在精巢中表达,为了验证EsDMRT-like蛋白是否在中华绒螯蟹精巢中特异表达及其功能,根据中华绒螯蟹EsDmrt-like基因序列,构建重组质粒pET-32a-EsDmrt-like,转化大肠杆菌BL21,经融合表达和SDS-PAGE分析表明,融合蛋白主要以包涵体形式存在,分子量约为46ku.利用Ni柱亲和纯化融合蛋白免疫家兔,制备获得EsDMRT-like多克隆抗体.Western-blotting检测表明该抗体既能特异地识别重组蛋白,又能特异识别精巢中EsDMRT-like蛋白,并且该抗体仅在精巢中检测到EsDMRT-like蛋白的表达,分子量约为52 ku,为预期单体分子量的二倍.Western-blotting检测变性后精巢总蛋白,该抗体能识别52和34 ku两条条带,证明了二聚体的存在.这一结果暗示EsDMRT-like可能通过形成二聚体形式调控中华绒螯蟹精巢发育.     

鲤疱疹病毒3型ORF136基因编码蛋白多克隆抗体的制备与鉴定

为了对鲤疱疹病毒3型(Cyprinid herpesvirus 3,CyHV-3)ORF136基因编码蛋白进行功能研究和血清学诊断,本实验通过对ORF136基因推导的第31~157位氨基酸序列进行PCR扩增,并与原核载体pET-32a(+)连接,转化至大肠杆菌Rosetta(DE3)感受态后进行IPTG诱导表达,将纯化后的重组蛋白免疫新西兰白兔(Oryctolagus cuniculus)以制备ORF136多克隆抗体,运用Western blot和间接免疫荧光技术对抗体进行鉴定。结果表明,重组融合表达蛋白大小与预期一致,约为35 kD,且主要分布在包涵体中。Western blot分析显示,免疫兔后获得的纯化ORF136多克隆抗体能特异性识别纯化的CyHV-3和感染CyHV-3的KS细胞;间接免疫荧光分析进一步表明ORF136多抗能识别感染CyHV-3的KS细胞。ORF136多克隆抗体的制备为ORF136蛋白功能研究和CyHV-3血清学诊断方法的建立提供了重要基础。     

Isolation and partial characterization of Asian sea bass (Lates calcarifer) Vitellogenin

A study was conducted to isolate, partial characterize Asian sea bass (Lates calcarifer) vitellogenin (vtg). Two-year-old juvenile L. calcarifer (n = 10) were given three intraperitoneal injections of 17-β estradiol (E₂) at a dose of 2 mg/kg body weight to induce vitellogenesis. Blood was collected 3 days after the last injection, and plasma was purified through gel filtration chromatography. A broad single symmetrical peak consisting of vtg molecule was produced. Protein concentration was 0.059 mg/ml as determined by Bradfrod assay using bovine serum albumin as a standard. The protein appeared as one circulating form in Native PAGE considering the dimeric form of putative vtg with molecular weight of 545 kDa. In SDS-PAGE under reducing conditions, two major bands appeared at 232.86 and 118.80 kDa and minor bands at 100.60, 85.80 and 39.92 kDa, respectively. The purified vtg was used to generate a polyclonal antibody, and the specificity of antibody was assessed by Western blot analysis. Two major bands were immunoreacted, but no cross-reactivity was observed with plasma from non-induced males. The protein was characterized as phosphoglycolipoprotein as it positively stained for the presence of lipid, phosphorus and carbohydrate using Sudan Black B, methyl green and periodic acid/Schiff reagent solution, respectively. The amino acid composition was analyzed by high sensitivity amino acid analysis that showed high percentage of non-polar amino acids (~48 %). The results suggest the potential utilization of vtg as a basis tool to further study about reproductive physiology of this important economical species.     

建鲤组织蛋白酶L 的原核表达、纯化鉴定及多克隆抗体的制备

对原核表达的重组建鲤组织蛋白酶L(Cathepsin L,CAT L)蛋白进行尿素洗涤和Ni-NTA亲和层析纯化,该目的蛋白经300 mmol/L咪唑洗脱为单一峰,SDS-PAGE结合TSK-GEL G2000SWxl凝胶过滤高效液相色谱分析表明重组CAT L获得了高度纯化,分子量约28 k D,纯度超过95%。Z-Phe-Arg-MCA底物测活法显示该重组CAT L表现为半胱氨酸蛋白酶活性,能与其内源抑制因子Cystatin以1︰1的摩尔比结合,具有生物学活性。以纯化的重组CAT L蛋白免疫Balb/C小鼠获得抗血清,经ELISA法检测获得的CAT L抗血清效价高于1︰512000;Western blotting鉴定结果表明该抗体具有良好的特异性,能够识别原核表达的重组CAT L蛋白。免疫组织化学分析结果表明,该抗体还能识别建鲤小肠、肝胰脏、脾、背肌和心肌组织表达的内源性CAT L蛋白。因此可利用该抗体从蛋白水平检测CAT L在鱼类不同组织中的表达和分布情况。     

尼罗罗非鱼无乳链球菌Sip蛋白乳酸菌活载体口服疫苗的研制及其免疫效果

链球菌病是威胁我国罗非鱼养殖产业健康发展的重要病害之一。为研制出免疫效果好、操作简便的罗非鱼链球菌病疫苗,本研究构建重组表达无乳链球菌Sip蛋白的穿梭质粒pNZ8124-Sip,通过酶切和测序验证后电转化乳酸乳球菌NZ9000,获得能够诱导重组表达无乳链球菌Sip蛋白的乳酸菌活菌载体疫苗。采用SPS-PAGE电泳摸索最佳诱导浓度和诱导时间以获得最大表达量,通过镍柱纯化目的蛋白并进行Western blot检测;利用不同浓度的重组乳酸菌活载体疫苗灌胃口服免疫尼罗罗非鱼,采用间接ELISA法测定免疫后血清抗体水平变化,通过人工腹腔注射感染无乳链球菌获得相对免疫保护率。研究结果显示,构建的重组乳酸乳球菌可通过nisin诱导表达大小为48 ku特异性蛋白,与目的蛋白大小一致;PAGE电泳显示,重组蛋白主要以可溶蛋白和包涵体2种形式存在,其中胞内可溶性蛋白浓度达7.65 mg/mL;诱导表达的最佳条件为100 ng/mL nisin诱导6 h;Western blot检测结果显示,诱导蛋白可与鼠抗His标签抗体特异性结合。口服免疫结果显示,中浓度组(2.24×10~(10) CFU/mL)和低浓度组(2.24×10~9 CFU/mL)免疫2次能够显著提高尼罗罗非鱼的血清抗体水平和抗无乳链球菌感染能力,中浓度免疫组的相对免疫保护率最高为41.0%。本研究可为罗非鱼链球菌病口服疫苗的研究奠定基础,具有广阔的应用前景。     

Expression, purification and characterization of yellow grouper Epinephelus awoara regulator of G protein signaling 16 protein

Regulators of G-protein signaling (RGS) proteins are a family of proteins, which accelerate GTPase-activity intrinsic to the alpha subunits of heterotrimeric G-proteins and play crucial roles in the physiological control of G-protein signaling. Here, yellow grouper RGS16 protein was expressed in Escherichia coli and purified by Ni–NTA affinity chromatography. The expression level of the fusion protein was up to 30% of the total cellular protein.Western blotting analysis showed that a band with the molecular mass of about 21 Kda was detected. The purified recombinant protein was used to prepare polyclonal antibody, and antiserum obtained was highly specific with the titer of over 1:32,000. Additionally, RGS16 protein was expressed in the Tn-5B1-4 insect cells. Western blotting analysis revealed that the expressed protein had immunoreactivity.