Prokaryotic expression, purification and characterization of nattokinase

Nattokinase is a fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy. In this study, nattokinase was purified from fermentation broth of a Bacillus subtilis strain by ammonium sulfate salting-out, gel filtration chromatography, and hydrophobic interaction chromatography with a purification fold of 5.2 and at a yield of 46.3%. The purified enzyme has molecular mass of 28 kDa and fibrinolytic activity of 4 580 U/mg. Since the concentration of nattokinase on fermentation broth was quite low, we cloned nattokinase gene from B. subtilis and expressed it in E. coli BL21 (DE3). Nattokinase was actively expressed in the recombinant strain. The yield of nattokinase was increased significantly, but the activity of the protein produced by recombinant strain was low.     

用Overlap-PCR法从Trichodermareesei QM9414基因组DNA中克隆并表达木聚糖酶Ⅲ

禾本科植物木聚糖酶在其成熟过程中需在细胞进入程序性死亡后 ,经蛋白水解酶多次剪切方显活性 ,常规蛋白质克隆表达系统无法表达这类酶。通过GenBank搜索获得一与之同族、结构相似的来源于T .reeseiQM94 1 4(ATCC2 6 92 1 )突变种PC 3 7菌株的xynⅢ。但该酶在T .reeseiQM9414中不表达 ,而在基因组中存在。通过overlap PCR法将 4个外显子分别克隆、测序 ,再连接测序 ,最终获得该基因的全长cDNA序列。将该基因连接到表达载体pETBlue-2上 ,并转化到TunerDE3表达菌株中 ,常规条件下可表达并有木聚糖酶活性显示。低温 (15℃)64h诱导显著提高可溶性xynⅢ酶活.     

Nuclear translocation of EGF receptor regulated by Epstein-Barr virus encoded latent membrane protein 1

Epstein-Barrvirus(EBV),oneoftheDNAon-cogenicviruses,iscloselyassociatedwiththegenesisofBurkitt抯lymphoma,undifferentiatednasopharyn-gealcarcinoma(NPC),Hodgkin抯disease,gastriccancerand,lungcancer,etc.[1].EBVencodedlatentmembraneprotein1(LMP1)isconsideredtobethemajoroncogenicproteinofEBVencodedproteins.Biologicallyspeaking,LMP1isanintegralmembraneproteincontaining386aminoacids.Thethreedo-mains(CTAR1,CTAR2,CTAR3)intheC-terminusofLMP1havebeenshowntoinitiatethesignalingproc-ess.The…     

Discovery of laryngeal carcinoma by serum proteomic pattern analysis

Laryngealcarcinomaisthemostcommonma-lignancyamongtheheadandneckcarcinomas.Theincidenceoflaryngealcanceraccountsforapproxi-mately5%ofmalignanttumorsinthepopulationofdevelopedcountries[1].Laryngealcarcinomahasthreesubtypes,whichincludesupraglottis,glottisandsub-glottis.Amongthem,glottisisthemostcommononeandaffectsmorethan50%ofallthelaryngealcancerpatients.Thoughlaryngoscopyisthemainstayforla-ryngealcancerdetectionbecauseofitsgeneralavail-ability,diagnosisisconfirmedbybiopsyofthepri-marylesion.E…     

X连锁隐性遗传性Marinesco-Sjögren综合征一家系五代11例报告

报告一个Marinesco-Sj?gren综合征(MSS)的大家系.通过家系调查和查体发现MSS患者11例,均为男性,已死亡7例,死亡年龄为15～20岁.患者均具有先天性白内障,智能发育迟滞、小脑性共济失调及骨骼畸形等征候.存活4例还伴有FSH水平增高,LH及T水平降低.结论认为该MSS家系为X连锁隐性遗传。 Abstract:A big family of Marinesco-Sjögren Syndrome with X-Linked recessive inheritance is reported in the paper.We discovered 11 Patients with MSS by family line inheritance and somatoscopy.These Patients were aged from 15 to 20 years old when they died with clinic feature of congenital cataract,mental retardation,cerebellar ataxia and skeleton deformity;4 patients survived,but showed high level of FSH plasme and low level of LH and T.is concluded that this is a MSS family X-Linked recessive inheritance.     

cDNA微阵列制作的优化

为了优化筛检cDNA微阵列中靶基因的最适长度、浓度及点样溶液的种类,设计持家基因beta actin和GAPDH RT-PCR 3对引物,产物长度在189～1 078 bp之间,以乙肝病毒DNA片段为阴性对照,扩增纯化后分别溶于3×SSC、50%DMSO及0.5mol/L碳酸盐缓冲液（pH=9.0）中,调整浓度分别为0.5μg/μL、1.0μg/μL和1.5μg/μL,比较上述不同条件的杂交结果。结果表明,杂交具有较好的特异性,阴性对照（乙肝病毒）和空白对照（点样溶液）均未见杂交信号;3种长度的同一靶基因杂交信号强度无明显差别（beta actin P=0.378;GAPDH P=0.866）;3种点样溶液中以50%DMSO杂交信号最好,较强且均匀一致(P=0.0001),其余2种差异不显著(P=0.142);3种浓度靶基因杂交信号差异不显著(P=0.648),浓度高者信号略强。短片段靶基因（200 bp左右）可获得与长片段靶基因（1 000 bp以上）一样较好的杂交信号,点样溶液以50%DMSO效果最好,靶基因浓度为0.5μg/μL时即可得到较好的杂交结果。 Abstract:To optimize and screen the most suitable target gene length,concentration and printing solution in cDNA microarray,housekeeping genes,such as beta actin and GAPDH,were selected as targets and hepatitis B virus gene as negative control.The RT-PCR primers that spanned at least one intron and whose products were at between 189 bp and 1 078 bp were designed with primer premier 5.0,so did the hepatitis B virus gene PCR primer.After polymerase chain reaction,the products were purified with ethanol and dissolved in 3×SSC,50% DMSO and 0.5mol/L carbonate buffer（pH=9.0）respectively.The concentrations of target genes were adjusted at 0.5μg/μL,1.0μg/μL and 1.5μg/μL.The hybridization signals had a good specificity.No signal showed in either negative control (HBV) or blank control (printing solution only).There was no significant difference in target gene lengths.The P value of beta actin (189 bp,491 bp,974 bp) and GAPDH (227 bp,552 bp,1 078 bp) was 0.378 and 0.866 respectively.There was no significant difference among concentrations（P=0.648）,too.However,the higher the concentration was,the stronger the signals would be.Among the three kinds of printing solution,50% DMSO was the best（P=0.0001）,while the other two had no difference by multi-comparison(P=0.142).The target gene at length between 200 bp and 1 000 bp has got the same hybridization signals.50%DMSO printing solution and the target gene concentration of 0.5μg/μl are suitable for good hybridization.     

Streptomyces hygroscopicus谷氨酰胺转胺酶酶原基因的鉴定及在大肠杆菌中的表达

[目的]鉴定来源于吸水链霉菌的谷氨酰胺转胺酶基因;研究其在大肠杆菌系统的克隆与表达;分析该酶与其同源酶的活性中心氨基酸序列.[方法]从本实验室筛选的吸水链霉菌(Streptomyces hygroscopicus;CCTCC M203062)发酵液中,分离纯化得到谷氨酰胺转胺酶酶原(pro-MTGase),测得N-端前十个氨基酸序列并与其它链霉菌来源的相应基因序列比较设计引物,扩增得到pro-MTGase 基因,将该基因插入到表达载体pET-20b( )信号肽pelB下游,构建分泌型表达载体pET/pro-MTG,并转化不同的大肠杆菌宿主BL21(DE3)和Rosetta(DE3)pLysS.[结果]获得了pro-MTGase的完整基因序列,多重碱基序列比对表明其与S.platensis和S.caniferus的pro-MTGase基因同源性高达92%.利用Rosetta(DE3)pLysS通过降温至24℃诱导策略,获得部分胞外表达的酶原.SDS-PAGE显示,胞外表达重组蛋白的分子量约为44kDa,与吸水链霉菌表达的天然酶原相符.诱导4 h后发酵液中的重组酶原经胰蛋白酶活化为成熟酶后测得最高酶活为0.24U/mL.[结论]该研究是对吸水链霉菌的谷氨酰胺转胺酶基因的首次报道,也是国内首次利用大肠杆菌实现pro-MTGase的胞外可溶性表达.     

关于草鱼肠炎微生态调节的研究

对健康草鱼肠道菌、患肺炎病草鱼肠道菌进行菌群分析,发现正常草鱼肠道中有气单胞菌(Aeromonas)、哈夫尼亚菌(Hafnia)、黄杆菌属(Flavobacterium)等多种菌,肠炎病鱼肠道中一般仅有气单胞菌的单一菌群。拮抗试验表明哈夫尼亚菌与气单胞菌之间有一定的拮抗性。回归试验表明哈夫尼亚菌无致病性,3种气单胞菌有致病性。微生态治疗试验表明哈夫尼亚菌对患肠炎病的草鱼肠道有促进肠道微生态平衡作用。     

鲤科四种鱼肠道菌群的相似性及其与系统进化的关系

在同一背景条件下,对草鱼、白链、团头鲂和鲤等4种鱼的肠道菌群中好氧性菌或兼性厌氧菌进行分析鉴定,得到4种鱼之间的肠道菌群相似值。用UPGMA和NJ法构建鲤科的雅罗鱼亚科,链亚科,Bian亚科和鲤亚科等4种亚科的系统演化树。不同亚科的鱼间菌群分布有较大差异。但雅罗鱼亚科和Bian亚科和菌群分布方面有极显的正相关性,哈夫尼亚菌属、致病杆菌属、气单胞菌属、柠檬酸菌属、假单胞菌属等在4种鱼肠道中均有分布,链球菌属,拉恩氏菌属、牙包杆菌属、葡萄球菌属等属于共生菌,雅罗鱼亚科和Bian亚科相邻邻接为一个共同接点,该接点与鲤亚科连接为一个接点,鲤亚科可能是最原始的类型。