马氏珠母贝α2-巨球蛋白受体结合区酵母双杂交系统的构建及自激活检测

【目的】构建马氏珠母贝(Pinctada martensii)酵母双杂交文库及α2-巨球蛋白受体结合区诱饵载体,检测其在酵母细胞中的自激活作用。【方法】利用CLONTECH SMART技术及酵母体内同源重组的方法构建马氏珠母贝酵母双杂交cDNA文库;将α2-巨球蛋白受体结合区克隆至pGADT7载体,并转化AH109酵母感受态细胞,检测自激活作用。【结果与结论】酵母双杂交文库容量为1.11×107克隆/mL,重组率大于90%;菌落PCR结果显示,插入片段长度均在500bp以上;诱饵质粒成功转化至AH109菌株,且无自激活性,符合文库构建指标,可用于文库筛选。     

草鱼呼肠孤病毒096vp7基因生物信息学分析及其酵母表达载体的构建

草鱼呼肠孤病毒（Grass carp reovirus, GCRV）是草鱼出血病的病原.从患病草鱼体内分离到一株草鱼呼肠孤病毒GCRV 096,经反转录PCR,克隆得GCRV 096 长度为855 bp 的vp7 基因,并分析该基因编码蛋白的特性.结果表明,同一基因型分离株VP7 蛋白间的差异不大,但不同基因型分离株VP7 蛋白间存在很大的差异.对GCRV 096 VP7 蛋白生物信息学分析表明,信号肽位点位于20 氨基酸处,且VP7 含有4个潜在的抗原决定簇.构建GCRV 096 vp7 基因的酵母表达载体pGBKT7-S10,并成功转化至酵母中.     

水产养殖用芽孢杆菌制剂的检测

通过平板菌落计数法检测编号为GD1、GD2、GD3、GD4、GD5、JS、JX、SD1和SD2等9种水产养殖用芽孢杆菌制剂的有效活菌数,结合形态、生理生化和16S rRNA基因序列分析对各制剂中分离的产芽孢细菌代表菌株进行鉴定。结果显示:除GD1、GD2和GD5制剂未标明芽孢数量外,其余制剂检测到的芽孢数量均低于或远低于标注数量;经鉴定,各制剂中最优势类型,GD2、GD3、GD5、SD1和SD2制剂为地衣芽孢杆菌(Bacillus licheniformis),GD4、JS和JX制剂为解淀粉芽孢杆菌(B.amyloliquefaciens),GD1制剂为枯草芽孢杆菌(B.subtilis)。除GD4、GD5和SD1制剂未标明具体菌种以及GD2的标注菌种与检测结果不一致外,其他制剂的标注菌种与被鉴定的优势类型芽孢杆菌基本一致。     

溶藻弧菌dtd基因的克隆及原核表达条件优化

根据NCBI数据库中已发表的溶藻弧菌(Vibrio alginolyticus)全基因组序列合成一对特异性引物,应用聚合酶链式反应(PCR技术)扩增溶藻弧菌HY9001菌株dtd基因,将其定向克隆到原核表达载体pET-32a构建重组表达质粒pET-DTD,并对其进行诱导温度、诱导时间、诱导IPTG浓度等条件的优化,最后探究其纯化时最佳咪唑洗脱浓度。结果表明:DTD蛋白成功表达,且其以包涵体的形式存在,在诱导温度37℃、IPTG浓度0.1 mmol/L条件下诱导5 h表达量最高,纯化最佳咪唑洗脱浓度为150 mmol/L。     

口蹄疫病毒VP1植物表达载体的构建

P1 T重组质粒上含有口蹄疫病毒 (FMDV)GD10分离株的p1cDNA片段 ,以此为模板 ,用PCR方法扩增其中的VP1基因 ,获得大小约 6 40bp的片段。该片段用BglⅡ和BstEⅡ酶切消化后克隆至表达载体 pCAMBIA130 5 .2 ,转化EcoliTOP10感受态细胞。重组质粒经PCR、酶切及序列分析 ,证实VP1基因处于CaMV35S启动子控制 ,且读码框正确     

基于OSMAC策略的3株海洋真菌发酵条件筛选与优化

【目的】筛选可促进海洋真菌产生代谢产物更丰富的发酵条件,进一步挖掘菌株在抗阿尔茨海默症方面的潜力。【方法】基于OSMAC策略,分别选用马铃薯葡萄糖水(PDB)培养基、察氏培养基、大米培养基和葡萄糖/蛋白胨/酵母膏(GPY)培养基,并设置3种盐度(3、30和100),对Talaromyces sp. 001、Penicillium sp. 019和Aspergillus terreus ZN4-5-4等3株海洋真菌进行小规模发酵培养。通过观察真菌在培养基中的生长状态变化,并根据TLC指纹图谱、乙酰胆碱酯酶(AChE)抑制、1, 1-二苯基-2-三硝基苯肼(DPPH)自由基清除和卤虫致死活性筛选模型等技术手段筛选出代谢产物丰富、毒性小且AChE抑制活性与抗氧化活性产物丰富的发酵培养条件。【结果】菌株001的最优发酵条件是盐度3的GPY培养基,菌株019的最优发酵条件是盐度3的PDB培养基,菌株ZN4-5-4的最优发酵条件是盐度3的PDB培养基。【结论】OSMAC策略指导下,改变培养基条件对菌株产生次生代谢产物有显著影响。     

生物采油解堵剂中产蛋白酶菌株的筛选及培养条件优化

为研究生物酶采油解堵剂中产蛋白酶菌株的初、复筛选及培养条件优化,从大庆原油样品中筛选菌种,通过水解酪素的透明圈实验及福林酚测蛋白酶酶活的方法进行菌株的初、复筛选;以蛋白酶酶活为优化指标,采用单因素实验对筛选的产蛋白酶菌株的培养基及培养条件进行优化,优化最适培养基:可溶性淀粉为15g/L,蛋白胨为20g/L,酵母膏为20g/L,NaCl为1.0g/L,CaCl2为0.02g/L,Na2HPO4为0.2g/L,NaH2PO4为0.1g/L;在初始pH为6.0、接种量为5%(体积分数)、温度为31℃、摇床转速为160r/min的条件下,培养72h后,菌株的蛋白酶酶活为551.0U/mL,为复筛选菌株的蛋白酶酶活的22.92倍,即为菌株生长繁殖及代谢的最佳条件,能够获得更高的蛋白酶酶活,有利于后续实验的进行.结果表明:菌株产蛋白酶对原油作用效果为发酵液表面张力从作用前的56.2mN/m降低到作用后的30.5mN/m,表面张力显著降低,还有降解降黏原油等效果,具有一定的研究价值.     

新加坡石斑鱼虹彩病毒ORF162蛋白的表达、纯化及抗体制备

将新加坡石斑鱼虹彩病毒(Singapore grouper iridovirus,SGIV)的ORF162的开放式阅读框插入pET-32a表达载体T7启动子控制下的6-His·Tag编码基因上游,构建SGIVORF162原核表达质粒pET-ORF162。表达质粒转化入大肠杆菌BL21(DE3)菌株,经IPTG诱导,成功表达SGIV ORF162融合蛋白。对IPTG浓度、诱导温度、诱导时间等诱导表达条件进行优化后,确定在0.7mmol/LIPTG、16℃条件下诱导14h时可溶性SGIV ORF162重组蛋白占重组蛋白总量的95%。经镍琼脂糖凝胶纯化,获得纯度为90%以上的SGIV ORF162蛋白。用纯化的SGIV ORF162蛋白免疫小鼠,获得高效特异的SGIV ORF162多克隆抗体。     

融合蛋白IL6-OmpW的质粒构建及表达纯化

以重组质粒pMD18-T/IL6和pMD18-T/OmpW为模板,分别扩增红笛鲷IL-6基因和哈维氏弧菌外膜蛋白OmpW基因,运用PCR重叠延伸剪切技术,将IL-6和OmpW基因融合,将融合基因定向克隆到原核表达载体pET-32a(+),转化大肠杆菌BL21(DE3)感受态,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导融合蛋白高效表达,融合蛋白分子质量约为66.6 ku。优化后表达条件为温度37℃,IPTG浓度0.2 mmol·L-1,诱导时间5 h。用HisTrap HP亲和柱纯化重组蛋白,最佳咪唑洗脱浓度为400 mmol·L-1,纯化蛋白的质量浓度为480μg·mL-1。Western-blot分析显示,该融合蛋白可与鼠抗His-tag单克隆抗体发生特异反应,表明目的蛋白得以正确表达。     

尼罗罗非鱼Galectin-3基因的原核表达与条件优化

【目的】对Galectin-3蛋白进行纯化,优化诱导相关表达条件,为大量获取罗非鱼Galectin-3蛋白提供方案。【方法】根据NCBI上已公布的罗非鱼(Oreochromismossambicus)Galectin-3基因序列,设计多对带Eco RI和Xhol酶切位点的引物,筛选出良性扩增引物,对罗非鱼cDNA经进行聚合酶链式反应(PCR)扩增、限制性快切酶双酶切、T4连接酶连接等步骤,构建罗非鱼Galectin-3基因的原核表达质粒pGEX-4T-Galectin-3,将重组质粒导入大肠杆菌BL21中,进行Galectin-3重组蛋白的表达。并比较不同的诱导温度、诱导时间、IPTG浓度条件下的表达效果,对Galectin-3蛋白表达最优条件进行筛选。【结果】构建了罗非鱼Galectin-3原核表达质粒,进行Galectin-3重组蛋白后发现37℃下,0.4 mmol/L浓度的IPTG诱导5 h即可诱导Galectin-3重组蛋白高效表达。免疫印迹结果显示,经蛋白纯化柱纯化后的pGEX-4T-Galectin-3重组蛋白可与GST-Tag单克隆抗体发生特异性反应,进而表明表达的重组蛋白是罗非鱼的Galectin-3蛋白。【结论】本研究成功构建了罗非鱼Galectin-3基因的原核表达载体,并确定了Galectin-3融合蛋白最适的表达条件:37℃下,0.4 mmol/L浓度的IPTG诱导5 h。     

一株H_5亚型禽流感病毒血凝素基因的克隆与序列分析

根据GenBank中发表的H5亚型禽流感病毒HA基因序列设计2对引物,用RT-PCR方法从禽流感病毒广东分离病毒株(A/Chicken/Guangdong/1997)中扩增HA基因cDNA片段,并将其克隆至pMD-18T载体进行核苷酸序列测定。结果表明:用2对引物所扩增的片段大小分别约为1300 bp和800 bp,经序列拼接获得的HA基因cDNA长度约为1601 bp,编码533个氨基酸,与国内己发表的11个代表株的核苷酸和氨基酸序列同源性为分别为96.9%~99.9%和86.5%~93.0%;HA基因编码的氨基酸序列的系统进化树也表明A/Chicken/Guang-dong/1997、A/Goose/Huadong/01/2000、A/Ck/Hk/37.4/2002、A/Chicken/Zhoukou/2/02、A/Duck/Guangxi/53/2002、A/Duck/Fujian/01/2002等毒株处于同一进化枝,亲缘关系较近;而与A/Silly/Chicken/Hongkong/SF189/01株处于不同进化枝,亲缘关系较远。     

面包酵母悬浮液中添加脂溶性维生素对褶皱臂尾轮虫增殖率的影响

采用批量培育法研究了在面包酵母悬浮液中添加不同浓度的脂溶性维生素对褶皱臂尾轮虫（Ｂｒａｃｂｉｏｎｕｓｐｌｉｃａｔｉｌｉｓ）增殖率的影响。结果表明：面包酵母对褶皱臂尾轮虫的生长繁殖是有营养缺陷的。在面包酵母悬浮液中．当添加维生素Ａ为０．０２～５．００μｇ／ｍｌ时，随着添加浓度的增大，轮虫的种群增殖率Ｒ值也增大。以添加５．００μｇ／ｍｌ为最佳，第４天的Ｒ值达０．７５，是对照组的５．６倍。添加维生素Ｄ０．００５～０．２０μｇ／ｍｌ对轮虫的增殖有一定促进作用，以添加０．０５μｇ／ｍｌ为最佳，第７天的Ｒ值为０．４０７，是对照组的２．７倍。添加维生素Ｅ０．００１～０．０１μｇ／ｍｌ和添加维生素Ｋ０．０１～０．１０μｇ／ｍｌ时，第４天的Ｒ值与对照组无差异。     

Molecular Cloning and Expression ofPoIR2, a Novel Gene Involved in Immune Response in Japanese Flounder （Paralichthys olivaceus）

A novel immune-related gene was expressed in Japanese flounder (Paralichthys olivaceus) injected with Vibrio anguillarum. The complete cDNA contained a 169 bp 5’UTR, a 336 bp open reading frame (ORF) encoding 111 amino acids and a 556bp 3’UTR. Six exons and five introns were identified in the PoIR2 gene. Blastp similarity comparison showed its encoding protein had 50% similarity to Danio rerio neuromedin S (NMS), but further alignment indicated they did not have NMS C-terminal conservational signature domain. So it was not defined as an NMS homologue. Protein structure analysis indicated it had a 26aa signal peptide and was a secretory pathway protein. RT-PCR demonstrated that the expression of PoIR2 was quickly induced and drastically increased in liver, kidney, spleen, gills, intestine, heart, and skeletal muscle after infected with V. anguillarum. These results indicated that the PoIR2 might play some important role in Japanese flounder immune response system. This gene was named PoIR2 (P.olivaceus immune-related gene 2, GenBank accession number: EU224372). The mature PoIR2 peptide was expressed in BL21(DE3) pLysS using pET-32a(+) vector and a great part of the recombinant mature peptide existed as soluble type.     

Molecular identification,expression pattern,and in-vitro bioactivity analysis of insulin-like growth factor 2 in olive flounder Paralichthys olivaceus

Insulin-like growth factors (IGFs) are key regulators of development and growth. Here, we characterized the igf2 gene from olive flounder (Paralichthys olivaceus) and determined its temporal and spatial expression. We set up an in-vitro protein expression system in eukaryotic human embryonic kidney (HEK293T) cells and explored its effects on cell proliferation. The flounder igf2 cDNA contained a 648-bp open reading frame (ORF) encoding a protein of 215 amino acids (aa), which spanned the complete signal peptide (47 aa), mature peptide (70 aa), and E domain (98 aa). In adult flounder, igf2 mRNA was detected in all selected tissues. In early development, igf2 mRNA was detected throughout development from unfertilized eggs to hatching-stage embryos. In-situ hybridization analysis indicated that igf2 mRNA was specially expressed in the brain region, floor plate, hypochord, otic vesicle, and pectoral fin during embryogenesis. Western blotting analysis indicated that the soluble recombinant flounder IGF2 protein was successfully produced through eukaryotic expression in HEK293T cells. In addition, the recombinant IGF2 protein significantly promoted the proliferation of human cervical carcinoma (HeLa) and HEK293T cells. These results provide new information about the structural and functional conservation, expression patterns, and biological activity of the igf2 in teleosts.

     

红笛鲷重组激活基因rag1原核表达条件的优化及纯化

克隆编码红笛鲷(Lutjanus sanguineus)RAG1蛋白(recombination activating protein1)活性核心区的基因序列,并与pET-28a(+)载体连接,构建原核表达载体pET-28a-RAG1,将其转入大肠杆菌BL21(DE3)菌株,利用IPTG进行诱导表达。为提高融合蛋白的表达效率,运用传统的实验方法对诱导条件进行优化。SDS-PAGE分析表明,在37℃条件下,利用0.1 mmol/L IPTG诱导8 h后,RAG1重组融合蛋白的表达量最大,相对分子质量与预测值相符,该蛋白主要以包涵体形式高效表达,利用His Trap HP亲和柱使其得到进一步纯化;Western blot分析显示,该融合蛋白可与鼠抗His-tag单克隆抗体发生特异性结合,说明表达蛋白为目的蛋白。     

Cloning and nucleotide sequence of D-hydantoinase gene of marine polyphosphate-accumulating bacterium, Halomonas sp. YSR-3

Hydantoinase is involved in the production of optically pure amino acids from racemic 5-mono-substituted hydantoins. We measured the D-hydantoinase activity in marine Halomonas sp. YSR-3 and amplified the D-hydantoinase gene by PCR. The gene was inserted into vector pGM-T and transformed into E. coli TOP10. The positive transformants with the D-hydantoinase gene were sequenced. The sequenced fragment comprises 1 510 base pairs. The D-hydantoinase gene from YSR-3 is 77% similar to that from Pseudomonas entomophila L4 by searching against the NCBI databse. The protein product of the YSR-3 D-hydantoinase gene is 75%, 73%, and 70% similar to those from Pseudomonas fluorescens Pf-5, Marinomonas sp. MED121, and Burkholderia vietnamiensis G4, respectively. The difference of the D-hydantoinase gene between marine Halomonas sp. YSR-3 and other terrestrial organisms is distinct.     

Expression,purification, and subcellular localization of phospholipase C in Dunaliella salina

Plants possess effective mechanisms to respond quickly to the external environment.Rapid activation of phosphatidylinositol-specific phospholipase C(PLC)enzymes occurs after a stimulus.The PLC in Dunaliella salina play s important roles in growth and stress responses.However,the molecular basis of PLC action in D.salina remains little understood.To gain insight into the potential biological functions of this enzyme,we cloned a phospholipase C gene from D.salina in a previous study,named DsPLC(GenBank No.KF573428).Here,we present the prokaryotic expression,purification,and characterization of the DsPLC gene.The entire coding region of DsPLC was inserted into an expression vector pET32 a,and the DsPLC gene was successfully expressed in Escherichia coli.The DsPLC protein was purified and identified using a polyclonal antibody and western blotting.Expressing DsPLC fused with a green fluorescent protein(GFP)in onion showed that DsPLC-GFP was localized to the intracellular membrane.Quantitative real-time PCR analysis revealed that the relative expression of the DsPLC gene was induced significantly by 3.0-mol/L NaCl at 4 h.Our results support the importance of PLC enzymes in plant defense signaling.This study provides a basis for further functional studies of the DsPLC gene and for additional analysis of the potential roles of PLC enzymes in response to abiotic stress.     

Characterization of halophilic C50 carotenoid-producing archaea isolated from solar saltworks in Bohai Bay,China

Halophilic archaea comprise the majority of microorganisms found in hypersaline environments. C50 carotenoids accumulated in archaea cells are considered potential biotechnological products and possess a number of biological functions. Ten red colordes were isolated from brine water in a saltem crystaltizer pond of the Hangu Saltworks, China. 16S rRNA gene sequence analysis showed that the colonies belonged to the extremely halophilic archaea genera Halobacterium and Halorubrum. Two representative strains, Halobacterium strain SP-2 and Halorubrum strain SP-4, were selected for further study on the phenotypic characteristics and effects of salinity and pH on accumulation and composition of pigments in their cells. The archaeal strains were isolated and grown in a culture medium prepared by dissolving yeast extract （10 g/L） and acid-hydrolyzed casein （7.5 g/L） into brine water obtained from a I.ocal salt pond. Their optimum salinity and pH for growth were 250 and 7, respectively, although pigment accumulation （OD490/ mL broth） was highest at pH 8. In addition, at 150-300 salinity, increasing salinity resulted in decreasing pigment accumulation. Analysis of the UV-Vis spectrum, TLC and HLPC chromatograms showed that C50 carotenoid bacterioruberin is the major pigment in both strains.