Characterization of the genes encoding urease activity of Klebsiella pneumoniae

Abstract The genes encoding urease activity of Klebsiella pneumoniae were cloned and expressed in Escherichia coli . Transformants containing recombinant plasmids were selected by the antibiotic resistance phenotype and the production of urease in a Urease-test agar. Deletion derivatives of the parental recombinant plasmid were construced, and the relative position of six genes, necessary for urease production, was determined. Using a colorimetric assay it was demonstrated that some of the transformants exhibited ureolytic activity up to six-times greater than that of the original K pneumoniae isolate. Dot-blot DNA hybridization analysis revealed that the urease gene cluster of K. pneumoniae possesses no significant homology with those of Proteus species and Morganella morganii .     

k-卡拉胶酶CgkX催化区的重组表达菌株筛选和发酵优化

目的：CgkX 是来自QY203 的一种高活性、高稳定性的k-卡拉胶酶,本文旨在构建CgkX 催化结构域（CgkX-CM）重组表达菌株,并对发酵条件进行优化,提高CgkX-CM 的产量。方法：在分析k- 卡拉胶酶CgkX-CM基因序列的基础上,构建多种CgkX-CM 表达载体,在大肠杆菌BL21（DE3）中进行重组表达,通过酶活测定筛选其中产量最高的表达菌株,并优化发酵起始pH、诱导温度、IPTG浓度、装液量以及甘氨酸浓度等因素。结果：构建了5 种重组表达菌株,其中BL21(DE3)/pET22b-CgkX-CM 酶产量是其他重组菌株的7.4-11.6 倍。确定了该菌株最佳发酵条件为：培养基含1 g·L-1甘氨酸（起始pH 7.5）,装液量为75 mL,0.1 mmol·L-1 IPTG 20 ℃诱导28 h,酶产量是优化前的7.3 倍。结论：经过重组表达载体筛选和发酵优化,CgkX-CM产量大幅度提高,为今后比较CgkX 全长及其催化区域的酶学性质,确定C 端Big_2 结构域对CgkX的作用奠定了基础。     

蓄势待发的印度生物技术产业

构建的重组表达质粒pPIC9K-HSA-IFNα-2b经限制性内切酶SalI酶切线性化后电转化巴斯德毕赤酵母菌（P.Pastoris）SMD1168,将阳性转化子用甲醇诱导表达并进行PCR鉴定和Mut表型鉴定,再用Western-Blot法及Wish细胞-VSV病毒系统来鉴定表达蛋白的免疫原性及生物活性,同时,在摇瓶培养条件下,初步研究了甲醇诱导浓度及菌体诱导的起始OD值两因素对目的蛋白表达量的影响,本研究成功表达出具有高生物学活性的白蛋白融合干扰素蛋白（HSA-IFNα-2b）,对摇瓶发酵表达条件的初步摸索也为进一步在大规模发酵中提高目的蛋白在P.Pastoris的表达水平提供一个参考依据。     

心脉隆注射液联合重组人脑钠肽治疗慢性心衰患者的临床观察

目的:探讨心脉隆注射液联合重组人脑钠肽治疗慢性心衰患者的临床效果及对患者心功能、血清N端脑钠肽前体(N-terminal pro b-type natriuretic peptide, NT-proBNP)、胶原、一氧化氮(Nitric oxide, NO)、内皮素-1(endothelin-1, ET-1)水平的影响。方法:选择2015年1月至2017年5月在我院治疗的慢性心衰患者80例,根据随机数字法将其分为对照组和观察组。对照组采用重组人脑钠肽静脉治疗,观察组在此基础上加用心脉隆注射液,治疗14天。观察和比较两组患者治疗前后的心功能指标、血清NT-proBNP、胶原、NO、ET-1水平的变化、临床疗效及不良反应的发生情况。结果:治疗后,观察组总有效率为97.5%,显著高于对照组(82.5%,P0.05)。治疗前,两组左心射血分数(left ventricular ejection fraction, LVEF)、每搏输出量(SV, Stroke volume)、心脏指数(CI, Cardiac index)、心率(HR, heart rate)、血清NT-proBNP、NO、ET-1、胶原水平比较差异均无统计学意义(P0.05);治疗后,两组LVEF、SV、CI、NO均较治疗前明显升高(P0.05),血清NT-proBNP、ET-1、胶原水平均较治疗前明显下降(P0.05),且观察组治疗前后各指标的变化幅度均显著高于对照组(P0.05)。在治疗过程中,对照组出现1例低血压,2例头疼,1例恶心,不良反应发生率为10%;观察组出现1例头痛,不良反应发生率为2.5%,但两组不良反应发生率比较差异无统计学意义(P0.05)。结论:心脉隆注射液联合重组人脑钠肽治疗慢性心衰的临床疗效显著优于重组人脑钠肽静脉治疗,其可有效降低患者血清NT-proBNP、胶原、ET-1的水平并升高血清NO的水平、改善患者心功能,且安全性更高。     

Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol

The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the enzyme was 7.5, while the low temperature optimum at 37°C was the property that differed significantly from the majority of the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose.     

重组人骨保护素对聚乙烯颗粒刺激的人工关节置换术患者外周血IL-6和TNF-α表达的影响

目的：探讨重组人骨保护素（rhOPG）对聚乙烯颗粒刺激的人工关节置换术患者外周血IL一6和TNF一0l表达的影响,分析rhOPG防止关节置换术后假体无菌性松动的应用价值。方法：收集2009年1月-2011年3月我院收治的人工关节置换术患者的外周血清60份,采用梯度离心法分离出单个核细胞,并将其随机分成5组,每组12份。对照组：单个核细胞,模型组：单个核细胞＋1X109／mL聚乙烯颗粒,低剂量组：单个核细胞＋1×109／mLL聚乙烯颗粒＋10ng／mLrhOPG,中剂量组：单个核细胞＋1X109／mL聚乙烯颗粒＋100ng／mLrhOPG,高剂量组：单个核细胞＋l×109／mL聚乙烯颗粒＋1000ng／mLrhOPG。通过酶联免疫酶联免疫法（ELISA）＇~4定各组细胞上清液中白介素-6（IL-6）和肿瘤坏死因子（TNF-α）的含量,并进行组间比较。结果i（1）模型组、低剂量组、中剂量组、高剂量组的IL-6、TNF-α．含量均显著高于对照组（P〈0．05）。（2）低剂量组、中剂量组、高剂量纽的IL-6、TNF—α浓度均较模型组低（P〈0．os）。（3）随着rhOPG浓度的增加,IL-6和TNF．仪含量逐渐降低,组间比较差异有统计学意义（P〈0．05）。结论irhOPG可抑制聚乙烯颗粒刺激的人工关节置换术患者外周血1L-6和TNF—α．的表达,可有助于防治人工关节置换术后的无菌性关节松动。关键词：人工关节置换术;重组人骨保护素;假体松动;白细胞介素-6;肿瘤坏死N子     

肠溶微囊式重组鲤生长激素促生长剂的研制

采用草鱼生长激素夹心式酶联免疫吸附测定法和酶联免疫吸附受体测定法,研制肠溶微囊式重组鲤生长激素（rcGH)促生长剂,结果表明,肠溶树脂Ⅰ和Ⅱ溶解的pH阈值分别为6.0和6.6,当大于阈值时,肠溶微囊Ⅰ和Ⅱ内rcGH的释放率随pH值升高和时间延长而逐渐升高,直至rcGH完全释放,投喂胡子鲶（Clarias batra-chus)含肠溶微囊Ⅰ（内含rcGH0后,血清rcGH水平15h后开始升高,24h时达最大值,随后呈下降趋势,48h后血清中检测不到rcGH;而投喂含rcGH的药饵（1mgrcGH/g饵料）,6-60h在胡子鲶血清中均未检测到rcGH。说明肠溶微囊Ⅰ对rcGH起一定保护作用,肠溶微囊Ⅰ和Ⅱ具有抗酸肠溶胶囊的特点和作用。     

Expression of the cloned genes encoding the putrescine biosynthetic enzymes and methionine adenosyltransferase of Escherichia coli (speA, speB, speC and metK)

The speA, speB and speC genes, which code for arginine decarboxylase (ADCase), agmatine ureohydrolase (AUHase) and ornithine decarboxylase (ODCase), respectively, and the metK gene, which encodes methionine adenosyltransferase (MATase), have been cloned. The genes were isolated from hybrid ColE1 plasmids of the Clarke-Carbon collection and were ligated into plasmid pBR322. Escherichia coli strains transformed with the recombinant plasmids exhibit a 7- to 17-fold overproduction of the various enzymes, as estimated from increases in the specific activities of the enzymes assayed in crude extracts. Minicells bearing the pBR322 hybrid plasmids and labeled with radioactive lysine synthesize radiolabeled proteins with M_rs corresponding to those reported for purified ODCase, ADCase and MATase. Restriction enzyme analysis of the plasmids, combined with measurements of specific activities of the enzymes in crude extracts of cells bearing recombinant plasmids, clarified the relative position of speA and speB. The gene order in the 62- to 64-min region is serA speB speA metK speC glc.     

重组人促红素联合多糖铁复合物胶囊对全膝关节置换术患者血液指标、膝关节功能及生活质量的影响

摘要 目的：探讨重组人促红素联合多糖铁复合物胶囊对全膝关节置换术患者血液指标、膝关节功能及生活质量的影响。方法：选取我院2019年2月至2022年2月期间收治的150例全膝关节置换术患者，应用随机数字表法分为对照组（常规围术期处理，n=75）与研究组（重组人促红素+铁剂，n=75）。对比两组临床指标、血液指标、膝关节功能及生活质量。结果：研究组的失血量和输血量少于对照组，住院时间短于对照组（P<0.05）。两组治疗后平均红细胞体积（MCV）、血清铁蛋白（SF）、血红蛋白（Hb）及平均血红蛋白浓度（MCHC）均较治疗前升高，且研究组治疗后的MCV、SF、Hb、MCHC高于对照组（P<0.05）。两组治疗后膝关节Lequesne指数评分、膝关节WOMAC量表评分均较治疗前下降，且研究组治疗后的膝关节Lequesne指数评分、膝关节WOMAC量表评分低于对照组（P<0.05）。两组治疗后心理功能、物质生活、躯体功能以及社会功能均较治疗前升高，且研究组治疗后的心理功能、物质生活、躯体功能以及社会功能评分高于对照组（P<0.05）。结论：重组人促红素联合多糖铁复合物胶囊用于全膝关节置换术患者，可提升MCV、SF、Hb、MCHC水平，促进膝关节功能及生活质量改善。