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1.
Phycoerythrin (PE) is one of the most important proteins involved in light capturing during photosynthesis in red algae. Its
potential biological activities had gained wide concerns. In the present study, tumor cytotoxic and hydroxyl radical assay
were preformed to detect the bioactivity of recombinant PE. Recombinant plasmids pGEX-PE and pBGL were transformed into E. coli BL21 to make two recombinant strains BEX (pGEX-PE) and BGL (pBGL). PE expressing in BEX (pGEX-PE) was validated by SDS-PAGE
and Western blotting analysis. SDS-PAGE analysis indicated that the PE-GST fusion protein was mostly inclusion bodies. Specific
expression of PE was confirmed by Western blotting analysis. The recombinant E. coli BEX (pGEX-PE) cells were collected and sonicated. The supernatants were reserved for the tumor cytotoxic experiments. The
result of tumor cytotoxic assay indicated that the supernatants containing PE had the activity of inhibiting the growth of
Hela cells and with the increase of protein concentration, the inhibiting rate increased from 37.31% to 63.26%, which showed
significant difference from the control. Hydroxyl radical scavenging effect was tested with supernatants of BEX (pGEX-PE)
and BGL (pBGL) cell lysates treated with sonication and heating. For the sonication samples, the scavenging rates of the supernatants
of BEX (pGEX-PE) and BGL (pBGL) cell lysates were significantly higher than the negative control BL21(pGEX-4T) (P<0.02), and the scavenging rates increased slowly following the increase of the protein content. For the heating samples,
except for the 0.2 mg mL−1 BGL (pBGL) products, the scavenging effects of the supernatants of BEX (pGEX-PE) and BGL (pBGL) cell lysates were stronger
than that of negative control BL21(pGEX-4T). However, the effect intensity was not positively correlated with the increase
of the protein concentration. Though a partially decreased hydroxyl radical scavenging activity was led by heating, the biological
activity was still retained and conspicuous. This research showed that phycoerythrin protein expressing in E. coli has the potential medical and sanitarian value. 相似文献
2.
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。 相似文献
3.
为探究溶藻弧菌(Vibrio alginolyticus)HY9901血红素结合蛋白HutB作为疫苗候选抗原的可能性,采用PCR方法扩增V.alginolyticus HY9901 hutB基因全长序列,克隆到pMD18-T载体,经双酶切、连接后,定向插入到pET-28a(+)中,构建原核表达载体pET-HutB,转入大肠杆菌BL21(DE3)中进行IPTG诱导表达。SDS-PAGE验证后,优化诱导表达条件和纯化条件,并进行Western-blot分析。结果表明,HutB蛋白在E.coli中诱导表达的最优条件:0.4 mmol/L IPTG,37℃诱导4h,表达的蛋白分子量与预期大小相符,主要以包涵体的形式存在,300 mmol/L咪唑洗脱时效果最佳,能与鼠抗His-tag单抗特异性结合。 相似文献
4.
An open reading frame (lcn61) of iymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector.Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene. 相似文献
5.
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。 相似文献
6.
β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion body.After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded AgaB showed a high specific hydrolysis activity of about 1600 units/mg protein. Forty milligrams of refolded pure protein were obtained from 1L of culture. 相似文献
7.
Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp (Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK’s (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future. 相似文献
8.
溶藻弧菌asp基因在大肠杆菌中表达活性研究及条件优化 总被引：1，自引：0，他引：1
为进一步研究溶藻弧菌碱性丝氨酸蛋白酶(ASP)蛋白生物学活性和功能,将构建的pET23d-asp在大肠杆菌BL21(DE3)中表达,对表达产物进行纯化分析,并优化表达条件。结果表明:在咪唑洗脱缓冲液浓度为100 mmol/L时,表达的可溶性ASP被大量洗脱,纯化的ASP相对分子质量约为42 000,具有蛋白活性;在诱导温度28℃、异丙基-β-D硫代半乳糖苷(Isopropyl-β-D-thiogalactopyranoside,IPTG)浓度1 mmol/L及诱导时间16 h时,表达的活性ASP蛋白最多。 相似文献
9.
Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the prote... 相似文献
10.
比较研究了水浸提、盐液浸提及超声辅助盐液浸提的近江牡蛎糖蛋白粗提物对羟自由基、DPPH自由基、超氧阴离子自由基的清除效果。结果表明:水浸提、盐液浸提及超声辅助盐液浸提的糖蛋白中可溶性蛋白与总糖含量之比分别为1.83∶1、4.49∶1和2.51∶1;3种糖蛋白粗提物均具有一定的清除羟自由基、DPPH自由基和超氧阴离子自由基的能力;它们对羟自由基的清除能力高于对DPPH自由基和超氧阴离子自由基;盐液浸提物清除超氧阴离子的能力最弱;超声辅助盐液浸提的糖蛋白粗提物清除羟自由基、超氧阴离子自由基及DPPH自由基的能力比水浸提和盐液浸提的糖蛋白的能力强。 相似文献
11.
ZENG Mingyong XIAO Feng ZHAO Yuanhui LIU Zunying LI Bafang and DONG Shiyuan College of Food Science Engineering Ocean University of China Qingdao P. R. China 《中国海洋大学学报(英文版)》2007,6(3)
Gelatin from the sea cucumber(Paracaudina chinens var.) was hydrolyzed by bromelain and the hydrolysate was found to have a high free radical scavenging activity. The hydrolysate was fractionated through an ultrafiltration membrane with 5 kDa molecular weight cutoff(MWCO). The portion(less than 5 kDa) was further separated by Sephadex G-25. The active peak was col-lected and assayed for free radical scavenging activity. The scavenging rates for superoxide anion radicals(O2·-) and hydroxyl radi-cals(·OH) of the fraction with the highest activity were 29.02% and 75.41%,respectively. A rabbit liver mitochondrial free radical damage model was adopted to study the free radical scavenging activity of the fraction. The results showed that the sea cucumber gelatin hydrolysate can prevent the damage of rabbit liver and mitochondria. 相似文献
12.
通过克隆编码红笛鲷(Lutjanus sanguineus)非特异性毒性细胞受体蛋白-1(nonspecific cytotoxic cell receptor protein-1,NCCRP-1)成熟肽基因序列,然后与载体连接构建pET21a-NCCRP融合蛋白表达载体,将其转入大肠杆菌BL21进行诱导表达并优化条件。SDS-PAGE分析表明,在异丙基-β-D-硫代半乳糖苷(IPTG)浓度0.8 mmol/L、37℃条件下培养3 h后表达量最大,分子大小与预期值相符,融合蛋白主要以包涵体形式高效表达,通过HisTrap HP柱子使其得到进一步纯化;Western blot分析表明,该融合蛋白可与鼠抗His-tag单克隆抗体发生特异性结合,说明获得该表达产物。 相似文献
13.
ZENG Mingyong XIAO Feng ZHAO Yuanhui LIU Zunying LI Bafang DONG Shiyuan 《中国海洋大学学报(英文版)》2007,6(3):255-258
Gelatin from the sea cucumber （Paracaudina chinens var.） was hydrolyzed by bromelain and the hydrolysate was found to have a high free radical scavenging activity. The hydrolysate was fractionated through an ultrafiltration membrane with 5 kDa molecular weight cutoff （MWCO）. The portion （less than 5 kDa） was further separated by Sephadex G-25. The active peak was collected and assayed for free radical scavenging activity. The scavenging rates for superoxide anion radicals （O2·^-） and hydroxyl radicals （·OH） of the fraction with the highest activity were 29.02% and 75.41%, respectively. A rabbit liver mitochondrial free radical damage model was adopted to study the free radical scavenging activity of the fraction. The results showed that the sea cucumber gelatin hydrolysate can prevent the damage of rabbit liver and mitochondria. 相似文献
14.
Xiaolin Chen Shengfeng Yang Ronge Xing Huahua Yu Song Liu Pengcheng Li 《中国海洋大学学报(英文版)》2012,11(2):249-252
In this paper, studies were carried out to extract astaxanthin from discharged wastewater during the production of chitin and to reveal the scavenging effect of the obtained pigment on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical. Different ratios of dichloromethane/methanol (V/V) were used to extract astaxanthin. When the ratio of dichloromethane/methanol was 2:8 and the ratio between the mixed organic solvent (dichloromethane/methanol, 2:8, V/V) and wastewater was 1:1, the highest yield of pigment was obtained (8.4 mg/ 50 mL). The concentration of free astaxanthin in the obtained pigment analyzed by HPLC was 30.02%. The obtained pigment possessed strong scavenging ability on DPPH radical and IC50 was 0.84mg/ml. 相似文献
15.
This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone (r-fGH) was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield (2.605 mgL-1) than that from soluble protein (1.964 mgL-1). Of the tested recovery methods, addition of renaturing buffer (pH 8.5) into denatured inclusion body yielded the best recovery rate (17.9%). This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture. 相似文献
16.
Din Siti Sabariah Chew Kit Wayne Chang Yu-Kaung Show Pau Loke Phang Siew Moi Juan Joon Ching 《中国海洋湖沼学报》2019,37(3):871-880
The effect of sonication pretreatment condition on Eucheuma cottonii and Gelidium amansii seaweed towards agar extraction wae studied. Four parameters were changed during sonication to investigate the effects on agar yield and quality. These parameters include the time interval, concentration ratio, frequency, and intensity. The highest amount of agar extracted from Eucheuma cottonii species could be obtained from the time interval of 30 min, seaweed weight to solvent volume ratio of 1:20, the frequency of 35 Hz, and the sonication power intensity of 30%. For Gelidium amansii species, the best agar yield also could be obtained from the time interval of 30 min, 1:20 of seaweed weight to water volume ratio, the frequency of 35 Hz, and power intensity of 30%. From the experiment, sonication pretreatment significantly influenced the yield and properties of extracted agar. The sonication with autoclaved seaweed produced agar containing less sulfate content, which is an excellent chemical property for gel electrophoresis applications.The gel strength of sonication with autoclaving for both seaweeds, Eucheuma and Gelidium species was the highest among those by sonication with direct heating, which proved that sonication pretreatment with autoclaving could enhance the physical properties of the agar. 相似文献
17.
Inorganic lead (Pb) is one of the most common environmental pollutants. Much evidence indicates that Pb exposure could directly affect fish growth and development. In this study, we investigated the cytotoxic effects of Pb on cytoskeletal protein stability at both protein and mRNA level in crucian carp Carassius auratus. Pb(NO3)2 treatment in concentration of 100 μmol/L resulted in decreased expression of both α- and β-tubulin but γ-tubulin as assayed with SDS-PAGE, Western Blot, and ELISA. In vivo and in vitro analyses on protein expression of tubulins are consistent. The effect of Pb on mRNA expression varied among different tissues. Our results suggest that cytotoxicity of Pb at protein translation level is stronger than at mRNA expression level. 相似文献
18.
《中国海洋湖沼学报》2017,(4)
Sea cucumber is a traditional nutritional food and medicinal resource with many bioactive components in China.Holothuria fuscogliva is a big sea cucumber with a rich of bioactive polysaccharides.To investigate the bioactivities of the polysaccharides from sea cucumber H.fuscogliva,we prepared the sulfated polysaccharides(HfP) from sea cucumber H.fuscogliva using a protease hydrolysis method.Antioxidant activities of HfP were investigated,including hydroxyl radical scavenging activity and superoxide radical scavenging activity.And,the anticoagulant activities of HfP were studied,including the activated partial thromboplastin time(APTT),prothrombin time(PT) and thrombin time(TT).The average molecular weight was 1 867.1 Da,with a sulfate content of 20.7%.In addition,the molar ratio of monosaccharide composition of HfP was Man:Rha:Glc A:Glc:Gal:Xyl:Fuc=0.083 6:0.437:0.134:0:1.182:0.748:1.It had a strong antioxidant activity,the hydroxyl and superoxide radical scavenging activity EC_(50) of HfP was 3.74 and 0.037 mg/mL,respectively.It also showed a good anticoagulant activity in our study.The APTT of HfP was much higher than that of heparin sodium,and the PT and TT of HfP was close to that of heparin sodium at a low concentration.Therefore,HfP shows a good antioxidant and anticoagulant activity and it may become a potential candidate of the natural antioxidant and anticoagulant and will have a good application future in health product or medicine industry. 相似文献
19.
Chia Shir Reen Chew Kit Wayne Show Pau Loke Sivakumar Manickam Ling Tau Chuan Tao Yang 《中国海洋湖沼学报》2019,37(3):898-908
Microalgae, a sustainable source of multi beneficial components has been discovered and could be utilised in pharmaceutical, bioenergy and food applications. This study aims to investigate the sugaring-out effect on the recovery of protein from wet green microalga, Chlorella sorokiniana CY1 which was assisted with sonication. A comparison of monosaccharides and disaccharides as one of the phaseforming constituents shows that the monosaccharides, glucose was the most suitable sugar in forming the phases with acetonitrile to enhance the production of protein(52% of protein). The protein productivity of microalgae was found to be significantly influenced by the volume ratio of both phases, as the yield of protein increased to 77%. The interval time between the sonication as well as the sonication modes were influencing the protein productivity as well. The optimum protein productivity was obtained with 10 s of resting time in between sonication. Pulse mode of sonication was suitable to break down the cell wall of microalgae compared to continuous mode as a lower protein yield was obtained with the application of continuous mode. The optimum condition for protein extraction were found as followed: 200 g/L glucose as bottom phase with volume ratio of I:1.25, 10 s of resting time for ultrasonication, 5 s of ultrasonication in pulse mode and 0.25 g of biomass weight. The high yield of protein about 81% could be obtained from microalgae which demonstrates the potential of this source and expected to play an important role in the future. 相似文献
20.
为探究溶藻弧菌(Vibrio alginolyticus)ZJ03株Ⅲ型分泌系统(Type III secretion system,T3SS)注射装置蛋白VscO作为疫苗候选抗原的可能性,根据GeneBank上登陆的溶藻弧菌VscO序列(NO.KJ179947),设计1对带酶切位点的特异性引物,PCR扩增vscO基因,序列分析结果显示,该基因全长462 bp,理论分子质量为18.430ku。将vscO基因定向插入原核表达载体pET-28a(+),构建重组表达质粒pET-vscO。用异丙基-β-D-硫代半乳糖苷(IPTG)诱导后,可在大肠杆菌(Escherichia coli)BL21(DE3)中表达分子质量约为22 ku的VscO融合蛋白,且该蛋白主要以包涵体形式存在。VscO蛋白表达和纯化的最优条件为:0.1 mmol/L IPTG、37℃条件下诱导4 h,咪唑洗脱浓度为400 mmol/L。用纯化后的融合蛋白免疫SPF级小鼠,获得高效多克隆抗体。Western-blotting结果表明,鼠抗VscO血清既能与重组VscO蛋白发生反应,也能与分离自溶藻弧菌约22 ku的天然蛋白发生反应,提示T3SS注射装置蛋白VscO可能是溶藻弧菌的重要保护性抗原之一。 相似文献