On the basis of the sequence and analysis of genome from the orange-spotted nervous necrosis virus（ OGNNV）, China strain, a pair of special primers were designed according to the nucleotide sequences of RNA2 from OG...On the basis of the sequence and analysis of genome from the orange-spotted nervous necrosis virus（ OGNNV）, China strain, a pair of special primers were designed according to the nucleotide sequences of RNA2 from OGNNV. The major capsid protein （ MCP）gene of OGNNV was cloned by means of reverse-transcriptase polymerase chain reaction （RT-PCR） and ligated into the pET32a expression plasmid. The MCP gene of OGNNV was 1 017 bases, encoded a protein of 338 amino acid with a molecular mass of 37.1 kDa. Recombinant protein with a molecular mass of 57.4 kDa was expressed in E. coli BL21 （DE3）. Vaccine was prepared from the recombinant protein expressed in recombinant cells. The juvenile orange-spotted groupers （8 cm in average length） were immunized by intraperitoneal injection. Group A was challenged with infected tissue filtrates 25 d post-vaccination. The mortality in the vaccined group （ A1,30% ） was a little higher than the unvaccined group （ B2, 27.8% ）. Group B was challenged after three vaccine injections. The mortality in the vaccined group （B1, 16.7% ） was lower than the unvaccined group （132, 27.8% ）, And the relative percentage survival （RPS） value of vaccined group, compared with the unvaccined group, was 40%. The anti-recombinant protein sera with a 1 ： 100 dilution were mixed with double volume of infected tissue filtrates and incubated at 4 ℃ for 12 h and then intramuscularly injected into the juvenile orange-spotted grouper. Treatment of infected tissue filtrates with anti-recombinant protein serum resulted in a significantly lower mortality of fish （ Group C1, mortality of 18.18% ）, compared with the fish （ Group C2, mortality of 40% ） which received infected tissue filtrates treated with control serum. Results implied the potential use of the capsid protein in immunization against OGNNV.展开更多
基金This work was supported by the National"863"Projects of China under contract No.2001AA601010the Natural Science Foundation of Jinan University,China under contract No.51204062.
文摘On the basis of the sequence and analysis of genome from the orange-spotted nervous necrosis virus（ OGNNV）, China strain, a pair of special primers were designed according to the nucleotide sequences of RNA2 from OGNNV. The major capsid protein （ MCP）gene of OGNNV was cloned by means of reverse-transcriptase polymerase chain reaction （RT-PCR） and ligated into the pET32a expression plasmid. The MCP gene of OGNNV was 1 017 bases, encoded a protein of 338 amino acid with a molecular mass of 37.1 kDa. Recombinant protein with a molecular mass of 57.4 kDa was expressed in E. coli BL21 （DE3）. Vaccine was prepared from the recombinant protein expressed in recombinant cells. The juvenile orange-spotted groupers （8 cm in average length） were immunized by intraperitoneal injection. Group A was challenged with infected tissue filtrates 25 d post-vaccination. The mortality in the vaccined group （ A1,30% ） was a little higher than the unvaccined group （ B2, 27.8% ）. Group B was challenged after three vaccine injections. The mortality in the vaccined group （B1, 16.7% ） was lower than the unvaccined group （132, 27.8% ）, And the relative percentage survival （RPS） value of vaccined group, compared with the unvaccined group, was 40%. The anti-recombinant protein sera with a 1 ： 100 dilution were mixed with double volume of infected tissue filtrates and incubated at 4 ℃ for 12 h and then intramuscularly injected into the juvenile orange-spotted grouper. Treatment of infected tissue filtrates with anti-recombinant protein serum resulted in a significantly lower mortality of fish （ Group C1, mortality of 18.18% ）, compared with the fish （ Group C2, mortality of 40% ） which received infected tissue filtrates treated with control serum. Results implied the potential use of the capsid protein in immunization against OGNNV.
基金This work was supported by the National Marine 863 Projects of China (Nos. 2003AA621010 2003AA621110) the NationalNatural Science Foundation of China (No. 39970586) Key Project of Scientific and Technological Research of the Ministry ofEducation (No
文摘研究斜带石斑鱼生长激素分泌及其 mRNA 表达的调控规律对于性别分化的控制、临床药物的选择,以及石斑鱼的增养殖等均具有重要的理论意义和实践意义。本文应用静态孵育系统, 采用放射免疫测定法和化学发光液相杂交实验, 研究GnRH 和 DA 对斜带石斑鱼 GH 分泌、GH mRNA 合成的调控作用。100 nmol/L sGnRH 作用斜带石斑鱼脑垂体碎片 1~24 h,明显促进GH的释放和GH mRNA的合成, 并具有时间依存性; 10 nmol/L^1 μmol/L sGnRH作用1 h 能明显促进斜带石斑鱼脑垂体释放GH,促进GH mRNA的合成, 表现出明显的剂量效应。100 nmol/L、1 μmol/L mGnRH 作用1 h 以一定的剂量依存方式促进 GH 的释放、促进 GH mRNA的合成,但 mGnRH 的效应比相应剂量的 sGnRH 的作用弱。APO 为 DA 受体的非选择性激动剂,不同剂量APO 对斜带石斑鱼脑垂体碎片的作用结果显示, 10 nmol/L^1 μmol/L APO 以剂量依存方式促进斜带石斑鱼脑垂体碎片释放GH、促进 GH mRNA的合成;1 μmol/L APO 作用 12 h 以上明显促进GH 的释放和 GH mRNA的合成,并随时间的延长而增加。与 sGnRH 对斜带石斑鱼 GH 释放、GH mRNA合成的作用相比, APO 的作用较弱。本文研究结果证实GnRH 和 DA能促进斜带石斑鱼脑垂体GH 释放和GH mRNA合成。
文摘白介素10(Interleukin-10,IL-10)是一个重要的多效细胞因子,主要作用是介导炎症反应和调控一些免疫细胞的分化和增殖。利用同源克隆和RACE-PCR技术获得全长为1 104 bp的斜带石斑鱼(Epinephelus coioides)IL-10基因。该基因ORF为564 bp,编码187个氨基酸,预测蛋白分子量为21.7 k D,等电点为5.74。氨基酸同源性比对及进化分析结果显示,斜带石斑鱼IL-10氨基酸序列和吉富罗非鱼(O.niloticus)同源性最高,达72.25%。利用双酶切及质粒重组技术构建IL-10原核表达载体,并转化大肠杆菌BL21(DE3)中诱导原核表达。SDS-PAGE分析显示,融合蛋白分子量为37.5 k D,与预期相符;在IPTG为0.02mmol/L,37℃诱导3 h,蛋白包涵体的表达量最大。
