Effects of recombinant human growth hormone on intestinal translocation of bacteria and endotoxin in rats with obstructive jaundice

Extrahepatic biliary obstruction promotes intestinal translocation of bacteria and endotoxin and this process is an important cause of morbidity and mortality in patients with jaundice. This study was undertaken to investigate the effect and mechanism of recombinant human growth hormone (rhGH) and to alleviate intestinal translocation of bacteria and endotoxin in murine obstructive jaundice. METHODS:A group of 42 Wistar rats were divided into 3 groups:sham operation (SO), bile duct ligation (BDL), and BDL and rhGH treatment (rhGH). By the end of the experiment,on day 7, the animals were killed, and their liver function and serum endotoxin were measured, bacterial cultures of the liver, kidney and mesenchymal lymph were made. Terminal ileum mucosa was observed under an electron microscope. RESULTS:Liver function was improved more significantly in the rhGH group than in the BDL group. The value of endotoxin in the rhGH group was 0.38±0.03 EU/ml, significantly lower than that in the BDL group (0.65±0.04 EU/ml, P【0.01), and similar to that in the SO group (0.30±0.02 EU/ml, P】0.05). The rate of bacteria translocation in the liver, kidney and mesenteric lymph was much higher in the BDL group than in other two groups. The rate of bacteria translocation in mesenteric lymph was 64.29%,significantly higher than that in the SO group and the rhGH group (P【0.05). There was no significant difference in bacteria translocation rate between the SO group and the rhGH group (P】0.05). Under an electron microscope , ileum mucosa epithelial cells in the BDL group were necrotic, and organelle were markedly metamorphic. In the rhGH group, ultrastructural changes were less evident or similar to those in the SO group. CONCLUSION:rhGH has significant protective effects on intestinal mucosa barrier in obstructive jaundice, and reduces intestinal translocation of bacteria and endotoxin.

Rapid and High-throughput Identification of Recombinant Bacteria with Mass Spectrometry Assay

Objective To construct a rapid and high-throughput assay for identifying recombinant bacteria based on mass spectrometry. Methods Matrix assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF MS） techniques were used to identify 12 recombinant proteins （10 of Yersinia pestis, 1 of Campylobacter jejuni and 1 of Helicobacter pylori）. A classification model for the various phase of recombinant bacteria was established, optimized and validated, using MALDI-TOF MS-CIinProTools system. The differences in the peptide mass spectra were analyzed by using Biotyper and FIexAnalysis softwares. Results Models of GA, SNN, and QC were established. After optimizing the parameters, the GA recognition model showed good classification capabilities： RC=100%, mean CVA=98.7% （the CVA was 96.4% in phase 1, 100% in phase 2, 98.4% in phase 3, and 100% in phase 4, respectively） and PPV=95}. This model can be used to classify the bacteria and their recombinant, which only requires 3.7x103 cells for analysis. The total time needed is only 10 min from protein extraction to reporting the result for one sample. Furthermore, this assay can automatically detect and test 96 samples concurrently. A total of 48 specific peaks （9, 16, 9, and 14 for the four stages, respectively） was found in the various phase of recombinant bacteria. Conclusion MALDI-TOF MS can be used as a fast, accurate, and high-throughput method to identify recombinant bacteria, which provide a new ideas not only for recombinant bacteria but also for the identification of mutant strains and bioterrorism pathogens.

Effects of Different Preservation Methods on Activity of Recombinant E. coli

[ Objective] To explore different preservation methods of recombinant E. coli and find out the optimal conditions for preservation. [ Method] The recombinant E. coli DH5cx transformed pcDNA.3 were respectively preserved at 4℃ and -70 ℃, and the activity was determined after dif- ferent time. [ Result] The number of living E. coll with high dilutions preserved at 4 ℃ was gradually increased within the first 7 d, peaked on Day 7, and then gradually decreased. The number of living E. coli, which were preserved in 8% glycerol at -70℃ when OD800 at 0.8, were significantly higher than that of other groups after different preservation time. [ Conclusion] The optimal storage time was 7 d for recombinant E. coli at 4 ℃. For preservation at -70 ℃, the bacteria should be in logarithmic growth phase and preserved in 8% glycerol.

猪表皮生长因子在乳酸乳球菌中的表达及其生物活性研究

【目的】构建表达猪表皮生长因子(pEGF)的乳酸乳球菌原核表达系统,并探究其对断奶仔猪腹泻的影响。【方法】以pUC57-SP45-LEISSTCDA-pEGF序列为模板,利用PCR扩增获得缺失LEISSTCDA片段的融合产物,将其与酶切pNZ8149表达载体连接后电转至乳酸乳球菌NZ3900,以LacF为筛选标记获得重组菌pEGF-NZ。用nisin诱导重组菌pEGF-NZ表达重组蛋白pEGF,并通过Tricine-SDS-PAGE检测其表达情况。改良实验室及工业发酵条件以提高pEGF表达量。利用猪回肠上皮细胞(IPEC-J2)增殖试验体外验证表达的pEGF生物活性。通过动物试验验证重组蛋白pEGF对小鼠及断奶仔猪腹泻的影响。【结果】试验成功构建重组菌pEGF-NZ,并分泌表达pEGF蛋白。体外试验结果显示,pEGF蛋白能促进细胞增殖。pEGF-NZ重组菌及其表达的pEGF蛋白能延缓小鼠腹泻发生时间,降低腹泻率,保护小鼠肠绒毛的完整性。与对照组相比,pEGF-NZ重组菌处理后,小鼠结肠中乳酸菌数显著增加(P<0.05),大肠杆菌数、肠球菌数显著降低(P<0.05),白细胞介素-1α(IL-1α)、IL-1β含量显著降低(P<0.05),IL-13含量显著升高(P<0.05)。饲粮中添加pEGF-NZ重组菌后,断奶仔猪腹泻情况明显缓解,平均日增重有升高趋势,但差异不显著(P>0.05)。【结论】pEGF-NZ重组菌及其分泌表达的pEGF能够修复肠道屏障,抑制病原菌黏附,调控肠道菌群,其主要通过下调部分促炎因子、上调抑炎因子表达发挥抑炎效果,从而保护断奶仔猪肠道健康,有效缓解腹泻。

重组Ⅰ型人源化胶原蛋白“福活因子”的生物合成及其结构表征研究

目的:筛选获得含胶原蛋白保守序列hCol.1基因的重组人源化Ⅰ型胶原蛋白高效表达基因工程菌和高产创新融合蛋白,并通过检测其理化特征验证表达产物。方法:将His6-SUMO-hCol.1重组基因大肠杆菌高密度发酵后经镍亲和层析和离子交换层析两步纯化浓缩获得重组蛋白,检测理化测试特征。结果:测序证明克隆基因与hCol.1序列高度一致,Blast比对蛋白序列与人Ⅰ型胶原蛋白高度同源,折叠结构与人Ⅲ型胶原蛋白高度类似。表观分子量55~60kD,产量36.4mg/L,纯度>95%。等电点检测、氨基酸组成和圆二色谱等实验结合高级结构预测分析,证明其理化性状与蛋白结构符合胶原蛋白特征。结论:成功将人Ⅰ型胶原蛋白保守功能结构域hCol.1与SUMO等肽段重组融合表达并命名为福活因子(FHTFF),筛选出基因工程菌pET21a(+)-FHTFF/BL21,纯化产物“福活因子”的理化特性、序列和结构与人Ⅰ、Ⅲ型胶原蛋白肽段高度相似,属于重组人源化胶原蛋白新生物材料。

重组人干扰素α-2b凝胶联合乳酸菌阴道胶囊对高危型人乳头瘤病毒感染患者阴道微生态的影响

目的探讨重组人干扰素α-2b凝胶联合乳酸菌阴道胶囊治疗高危型人乳头瘤病毒(HR-HPV)感染患者的临床疗效。方法选取2019年4月至2021年2月南昌三三四医院收治的88例HR-HPV感染者作为研究对象,按照随机数字表法分为观察组与对照组,每组44例。对照组采用重组人干扰素α-2b凝胶治疗,观察组在对照组基础上联合乳酸菌阴道胶囊治疗,比较两组临床疗效、阴道微生态恢复情况、免疫因子、不良反应发生情况及生命质量。结果观察组治疗总有效率为90.91%,高于对照组的70.45%,差异有统计学意义(P<0.05)。观察组阴道微生态正常或恢复率高于对照组,差异有统计学意义(P<0.05)。治疗后,观察组白细胞介素(IL)-4水平低于对照组,IL-12、干扰素γ(IFN-γ)水平均高于对照组,差异有统计学意义(P<0.05)。两组不良反应发生率比较差异无统计学意义。治疗后,两组健康调查简表(SF-36)评分均高于治疗前,且观察组高于对照组,差异有统计学意义(P<0.05)。结论重组人干扰素α-2b凝胶联合乳酸菌阴道胶囊治疗HR-HPV感染确切疗效,可有效促进患者阴道微生态平衡恢复,改善免疫因子水平,提升患者生命质量,且不良反应发生率较低,安全性较高。

乳酸菌阴道胶囊联合重组人干扰素α-2b凝胶治疗人乳头瘤病毒感染的效果探讨

目的:探讨乳酸菌阴道胶囊联合重组人干扰素α-2b凝胶治疗人乳头瘤病毒(HPV)感染患者的效果。方法:选取2022年6月—2023年6月湖南省职业病防治院妇产中心收治的HPV感染患者96例作为研究对象,随机数字表法分为对照组和观察组,各48例。对照组给予重组人干扰素α-2b凝胶治疗,观察组给予重组人干扰素α-2b凝胶联合乳酸菌阴道胶囊治疗。比较两组治疗效果、阴道微生态与不良反应发生情况。结果:观察组临床总有效率高于对照组,差异有统计学意义(P<0.05)。治疗前,两组患者阴道微生态情况比较,差异无统计学意义(P>0.05);治疗后,观察组Nugent评分、阴道分泌物pH值低于对照组,差异有统计学意义(P<0.05)。两组不良反应总发生率比较,差异无统计学意义(P>0.05)。结论:HPV感染患者给予乳酸菌阴道胶囊联合重组人干扰素α-2b凝胶治疗可以显著提高疗效,改善阴道微环境,不良反应较少,具有良好的药物安全性。

改性活性污泥处理5-羟甲基糠醛工业废水的研究

为了探讨5-羟甲基糠醛工业废水的处理方法。以乳酸杆菌基因(mob)组DNA为模板,运用PCR扩增到编码乳酸球菌的基因(a-als),并克隆到大肠杆菌表达载体PET28a上,构建克隆载体PET28a-moals,在E.coli TM109中成功表达。在限制性活性污泥中,通过发酵改性,使活性污泥细菌群中的目标菌改变成为优势菌,高效去除污染物,工业废水处理后回用,实现零排放。该方法是解决生物基产品工业废水的高效新方法,可以在化工、生物制药、功能性材料和其他相关行业推广应用。

Cloning of Bile Salt Hydrolase Gene and Its Expression in Lactic Acid Bacteria

According to the sequence of the bile salt hydrolase （BSH） gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase （BSH） gene was gotten from Bacillus bifidus ATCC 29521 by PCR. BSH gene was inserted into lactic acid bacteria expression vector pNZ8148 to construct the recombinant pNZ8148-BSH. The recombinant pNZ8148-BSH was transferred into lactic acid bacteria NZ9000 with electrotransformation method. And the recombinant which could express BSH protein was obtained. It was identified by SDS-PAGE electrophoresis and activity verification. The result could provide a rationale reference for expressing BSH in lactic acid bacteria.

乳酸菌基因组编辑技术研究进展

乳酸菌是一类重要的工业微生物,具有多种益生功能。目前关于其代谢功能改造、蛋白功能鉴定和挖掘优良功能基因等的研究主要依赖于传统的同源重组技术,但该技术效率低下,耗时且操作繁琐,严重制约了乳酸菌在基因水平的改造。因此建立稳定、高效的乳酸菌基因编辑方法,借助基因编辑技术挖掘功能基因、解析代谢途径、改良工业菌株等是十分有必要的。文章综述了乳酸菌基因编辑技术及其原理,并对这些技术在乳酸菌基因组上的应用现状和亟待解决的问题进行了分析总结,展望了乳酸菌基因组编辑技术的未来发展趋势,以期为乳酸菌功能基因鉴定及遗传改良提供参考。

鲢鱼重组cystatin C的抑菌活性及抑菌机理初探

cystatins是一类天然抗菌蛋白,广泛分布于鱼类加工废弃组织。采用滤纸片扩散法和二倍稀释法,评价了鲢鱼(Hypophthalmichthys molitrix)重组cystatin C(HmCystatin C)对6株常见的水产品腐败菌的抑菌活性;并以铜绿假单胞菌和腐生葡萄球菌为典型受试菌,测定HmCystatin C与菌体及其细胞壁成分脂多糖(lipopolysaccharides,LPS)和肽聚糖(peptidoglycan,PGN)的结合情况,及对受试菌生长曲线、细胞壁、细胞质膜通透性的影响,初步探讨其抑菌机制。结果表明,HmCystatin C对除枯草芽孢杆菌外的5株腐败菌均具抑制作用,对典型受试菌的最低抑菌浓度(minimum inhibitory concentration,MIC)及最低杀菌浓度(minimal bactericide concentration,MBC)均为3.5 mg/mL;HmCystatin C能与典型受试菌结合,并且对LPS和PGN表现出亲和性。此外典型受试菌的细胞壁和细胞质膜被破坏,胞内物质渗出。因此推测HmCystatin C可能首先吸附到菌体表面并与细胞壁成分结合,进而破坏细胞完整性,导致内容物外泄后菌体死亡。该结果为阐明鱼类cystatins抑菌机制奠定实验基础,也为综合利用鱼源cystatins提供理论参考。

重组人干扰素α-2b凝胶联合乳酸菌阴道胶囊治疗宫颈高危型人乳头瘤病毒持续感染的临床效果

目的探讨重组人干扰素α-2b凝胶联合乳酸菌阴道胶囊治疗宫颈高危型人乳头瘤病毒(HPV)持续感染的临床效果。方法选择2020年7月至2022年5月在酒泉市肃州区妇幼保健院妇科接受治疗的70例宫颈高危型HPV持续感染患者,采取随机数字表法分为研究组及常规组,每组各35例。常规组采取重组人干扰素α-2b凝胶治疗,研究组在常规组的基础上加用乳酸菌阴道胶囊,比较两组临床效果及阴道微生态情况。结果研究组治疗总有效率高于常规组(P<0.05);治疗后研究组阴道pH值、Nugent评分低于常规组(P<0.05)。结论重组人干扰素α-2b凝胶联合乳酸菌阴道胶囊治疗宫颈高危型HPV持续感染,能够提升治疗有效率,降低阴道pH值、Nugent评分。

Application of internal standard method in recombinant luminescent bacteria test

Mercury and its organic compounds have been of severe concern worldwide due to their damage to the ecosystem and human health. The development of effective and affordable technology to monitor and signal the presence of bioavailable mercury is an urgent need.The Mer gene is a mercury-responsive resistant gene, and a mercury-sensing recombinant luminescent bacterium using the Mer gene was constructed in this study. The mer operon from marine Pseudomonas putida strain SP1 was amplified and fused with prompterless lux CDABE in the p UCD615 plasmid within Escherichia coli cells, resulting in p THE30–E. coli.The recombinant strain showed high sensitivity and specificity. The detection limit of Hg^2＋was 5 nmol/L, and distinct luminescence could be detected in 30 min. Cd^2＋, Cu^2＋, Zn^2＋, Ca^2＋,Pb^2＋, Mg^2＋, Mn^2＋, and Al^3＋did not interfere with the detection over a range of 10-5–1 m M.Application of recombinant luminescent bacteria testing in environmental samples has been a controversial issue： especially for metal-sensing recombinant strains, false negatives caused by high cytotoxicity are one of the most important issues when applying recombinant luminescent bacteria in biomonitoring of heavy metals. In this study, by establishing an internal standard approach, the false negative problem was overcome;furthermore, the method can also help to estimate the suspected mercury concentration,which ensures high detection sensitivity of bioavailable Hg2＋.

Experimental study of human umbilica cord blood stromal cells transfected with recombinant adenoviral vector co-expressing VCAM-1

This work was funded from National Natural Science Foun-dation of China(No.39770335 and No.30070327)andChongqing Medical Key construction Foundation.Abstract:Ob-jective To establish the experimental foundation for furtherstudying the effect of VCAM-1 gene modified HUCBSCs

细菌基因工程杀虫剂研究进展

本文综述了在细菌基因工程杀虫菌构建过程中涉及到或可能涉及到的杀虫基因或因素的种类和特性 ,描述了工程菌构建的方法及策略 ,以及目前所获得的各类工程杀虫菌 ;讨论了杀虫工程菌的应用潜力和发展方向。

重组质粒对大肠杆菌HB101生长影响的微量热研究

用微量热方法研究了嗜麦芽假单胞菌AT18,受体菌大肠杆菌HB101,mel基因工程菌——大肠杆菌HB101/pWSY8和携带克隆载体pUC18质粒的大肠杆菌HB101等的生长代谢过程.实验结果从热化学和热动力学上阐明了细菌的生长速率常数与其所含质粒的大小呈负相关.探讨了低温处理对含不同质粒大肠杆菌生长的影响,发现低温处理对工程菌生长影响最大.

发酵五碳糖和六碳糖产乙醇的细菌研究进展

农作物废弃物中含有大量木质纤维素,经预处理或水解后能得到各种糖类的混合物,这些糖包括五碳糖和六碳糖(混合糖)。文中报道了国内外近20年来在利用细菌发酵混合糖产乙醇方面的研究进展。重点介绍了几种重组大肠杆菌Escherichia coli和运动发酵单胞菌Zymomonas mobilis利用混合糖产乙醇的特性。

基因工程重组乳酸菌防控动物疫病的研究进展

大多数传染性病原体通过黏膜表面进入宿主,因此除了传统诱导系统免疫的注射疫苗外,还应该开发能诱导黏膜免疫的疫苗,从而在黏膜侵入点建立第一道防线,阻止病原体侵入机体。乳酸菌是人和大多数动物肠道内常见的益生菌,具有营养、免疫、抗感染等作用。自20世纪90年代以来,乳酸菌便作为食品级载体用作外源基因的表达,其可刺激机体产生针对其表达的外源蛋白的免疫反应,因而基因工程重组乳酸菌可作为潜在的黏膜免疫候选疫苗。乳酸菌作为疫苗载体具有许多有吸引力的优势:使用方式简单、非侵入性(通过口腔或鼻内)、能接受基因修饰且遗传稳定、表达蛋白不需纯化、成本相对较低、安全性能最高、在体内长期存活并不断表达外源蛋白、单次接种即可产生持久的免疫应答等。目前,选择正常菌群的乳酸菌为载体,构建重组乳酸菌已在预防细菌、病毒、寄生虫等病原感染宿主方面迅速开展起来,将它们用于预防相应疫病模型中均已取得了较好的防治效果。作者就近5年来基因工程重组乳酸菌的制备及其在防控动物疫病方面的研究进展作一综述。

突变k-ras基因重组腺病毒的构建

目的 利用细菌内同源重组法构建含 1 2密码子点突变的k ras基因重组腺病毒。方法 用限制性内切酶kpnⅠ +xhoⅠ从载体pcDNA3 k ras1 2 (Val)中切出 1 2密码子点突变的k ras基因片断 ,亚克隆至经同样酶切的腺病毒穿梭质粒pAdTrack CMV中 ,形成转移质粒pAdTrack CMV/k ras 1 2 (Val) ,将之PmeⅠ酶切线性化后与腺病毒基因重组质粒pAdEasy 1共转化大肠杆菌BJ51 83 ,抽提经鉴定含目的基因的重组体质粒 ,PacⅠ酶切后用脂质体转染 2 93细胞 ,包装成重组体腺病毒Ad k ras1 2 (Val)。采用PCR方法对重组体腺病毒进行鉴定 ,利用穿梭质粒pAdTrack CMV中带有GFP报告基因 ,对病毒滴度和感染效率进行监测。结果 利用CaCl2 法由pAdTrack CMV/k ras 1 2 (Val)和pAdEasy 1共转化大肠杆菌BJ51 83 ,可获得 2 5%左右的阳性重组体细菌克隆。PCR检测表明重组腺病毒已含有目的基因 ,滴度为 1 .2× 1 0 1 2 pfu/ml。结论 细菌内同源重组法构建腺病毒相比于传统的细胞内同源重组法 ,具有成功率高、方法简便、快捷、实验周期短的优点 。

甜蛋白Monellin基因在大肠杆菌中的高效表达

根据已报道的单链monellin甜蛋白的氨基酸序列,采用细菌偏爱密码子,人工合成了全长294bp的monellin基因。插入到大肠杆菌表达载体pET_22b中,构建重组分泌型表达载体pETMO。经IPTG诱导pETMO所含有的甜蛋白基因可在大肠杆菌BL21(DE3)中高效表达,表达量占菌体可溶性蛋白的44·8%。且经纯化后测定其甜度是蔗糖的3000倍。得到的甜蛋白热稳性及耐酸性均比天然产物有所提高。