一株食源性多重耐药单核细胞增生李斯特菌的耐药性研究

本研究对食品样本中分离的一株多重耐药单核细胞增生李斯特菌进行耐药机制的探讨,以期对食源性单核细胞增生李斯特菌多重耐药现象的控制提供理论依据。本文通过聚合酶链式反应筛选耐药决定因子,质粒消除及自然转化实验对耐药决定因子进行定位及传播能力的探讨,最后通过传代实验验证该菌株多重耐药性传播的稳定性。结果表明,对检测到的多重耐药菌株LM78(耐受氯霉素、红霉素、链霉素、四环素、复方新诺明)进行相关耐药基因检测,检测到cat、erm B、tet S 3个耐药基因。质粒消除后MIC值下降到敏感范围,且该质粒可通过自然转化在不同菌属间传递,说明这些耐药基因存在于质粒上。该质粒在无抗生素选择压力下连续传代,仍具有较高稳定性。食源性致病菌多重耐药性有可能通过不同细菌种属间转移,进而由食物链向人类传播,对人类健康造成潜在的威胁。     

合成血红素加氧酶-1-shRNA载体的实验研究

根据Genebank提供的大鼠HO-1（Hmoxl,血红素加氧酶-1）基因序列,参照RNA干扰序列设计原则,设计针对HO—1的4个RNA干扰序列,定向克隆至表达载体pGPU6-GFP-Neo中,最后采用Baml-I I和PstI酶切凝胶电泳鉴定。鉴定结果显示成功合成了荷载HO-1-shRNA的表达载体。     

Continuous CHO cell cultures with improved recombinant protein productivity by using mannose as carbon source: Metabolic analysis and scale-up simulation

The replacement of glucose by mannose as a means to improve recombinant protein productivity was studied for the first time in continuous cultures of Chinese hamster ovary (CHO) cells producing human recombinant tissue plasminogen activator (t-PA). Steady-state operation at two hexose levels in the inlet (2.5 and 10 mM) allowed comparing the effect of sugar type and concentration on cell metabolism and t-PA production independently of changes in specific growth rates produced by different culture conditions. An increase in biomass concentration (15–20%) was observed when using mannose instead of glucose. Moreover, specific hexose consumption rates were 20–25% lower in mannose cultures whereas specific production rates of lactate, an undesirable by-product, were 25–35% lower than in glucose control cultures. The volumetric productivity of t-PA increased up to 30% in 10 mM mannose cultures, without affecting the sialylation levels of the protein. This increase is manly explained by the higher cell concentration, and represents a substantial improvement in the t-PA production process using glucose. Under this condition, the oxygen uptake rate and the specific oxygen consumption rate, both estimated by a stoichiometric analysis, were about 10% and 25% lower in mannose cultures, respectively. These differences lead to significant differences at larger scales, as it was estimated by simulating cell cultures at different bioreactor sizes (5–5000 L). By assuming a set of regular operating conditions in this kind of process, it was determined that mannose-based cultures could allow culturing CHO cells up to 3000 L compared to only 80 L in glucose cultures at the same conditions. These facts indicate that mannose cultures may have a significant advantage over glucose cultures not only in terms of volumetric productivity of the recombinant protein but also for their potential application in large-scale productive processes.     

Chitosan—A versatile semi-synthetic polymer in biomedical applications

This review outlines the new developments on chitosan-based bioapplications. Over the last decade, functional biomaterials research has developed new drug delivery systems and improved scaffolds for regenerative medicine that is currently one of the most rapidly growing fields in the life sciences. The aim is to restore or replace damaged body parts or lost organs by transplanting supportive scaffolds with appropriate cells that in combination with biomolecules generate new tissue. This is a highly interdisciplinary field that encompasses polymer synthesis and modification, cell culturing, gene therapy, stem cell research, therapeutic cloning and tissue engineering. In this regard, chitosan, as a biopolymer derived macromolecular compound, has a major involvement. Chitosan is a polyelectrolyte with reactive functional groups, gel-forming capability, high adsorption capacity and biodegradability. In addition, it is innately biocompatible and non-toxic to living tissues as well as having antibacterial, antifungal and antitumor activity. These features highlight the suitability and extensive applications that chitosan has in medicine. Micro/nanoparticles and hydrogels are widely used in the design of chitosan-based therapeuticsystems. The chemical structure and relevant biological properties of chitosan for regenerative medicine have been summarized as well as the methods for the preparation of controlled drug release devices and their applications.     

注射用重组人B淋巴细胞刺激因子受体-抗体融合蛋白对大鼠生育力及早期胚胎发育的毒性作用

目的观察注射用重组人B淋巴细胞刺激因子受体-抗体融合蛋白(RCT-18)对大鼠生育力及早期胚胎发育的毒性作用。方法将SD大鼠随机分为4组,分别经皮下注射RCT-18 129、37、11 mg/(kg.d)和0.9%NaCl注射液,每2 d给药1次。雄鼠连续给药5周、雌鼠连续给药2周后,按雌雄1∶1的比例合笼交配,合笼期为2周,计算交配率。雄鼠继续给药至交配成功,雌鼠继续给药至妊娠第6天。实验过程中观察动物的一般反应、体重及摄食量变化。确定雌鼠受精后处死雄鼠,解剖检查睾丸和附睾等生殖器官。于妊娠第15天解剖孕鼠,综合评价妊娠及胚胎形成和早期发育等情况。结果 RCT-18高、中、低剂量组雌性和雄性大鼠均未出现明显的母体毒性反应。各组大鼠各脏器均未见肉眼可见的异常。各剂量RCT-18对大鼠的交配率、妊娠率、生殖系统脏器重量和系数以及早期胚胎发育均无明显影响。结论RCT-18对SD大鼠的生育力及早期胚胎发育无明显毒性作用。     

人白细胞介素-6成熟肽基因重组及其在大肠杆菌中的表达

将PCR扩增的人白细胞介素-6（hIL-6）成熟肽基因与PJLA502表达载体重组，在大肠杆菌中高效表达出rhIL－6。经SDS－PAGE分析，rhIL－6表达量占菌体可溶性蛋白的40％以上。经免疫印迹证实，rhIL－6具有良好的抗原特异性。复性后，用MTT法测定IL－6依赖株7TD1细胞增殖活性，rhIL－6比活性达2．5×10~6u／mg蛋白。     

柱层析法纯化基因工程HBsAg中试工艺的研究

由乙肝病毒S基因转化的哺乳动物细胞培养收液，经过Butyl-S-SepharoseFF层析，DEAESepharoseFF层析和Sepharose4FF层析，可获HBsAg纯品。产品检定结果表明，PAGE和SDS-PAGE电泳纯度均合格，小牛血清残余量、细胞DNA残余量和动物免疫效力也均符合规定标准．HBsAg终收率达40％左右．在此工艺中，疏水作用柱层析的纯化效率比较高，可去除绝大部分杂蛋白和细胞DNA。     

重组腺病毒Ad-PML(NLS)-的构建及鉴定

目的构建携带PML(NLS-)基因的重组腺病毒,并观察其对白血病K562细胞增殖的影响。方法以质粒pCMV-HA-PML(NLS-)为模板,PCR扩增PML(NLS-)基因,与穿梭质粒pAdTrace-TO4连接,构建重组穿梭载体pAdTrace-TO4-PML(NLS-),经PmeⅠ酶切线性化后,转化入感受态BJ5183细菌,获得重组腺病毒质粒pAd-PML(NLS-),经PacⅠ酶切后,转染AD293细胞,获得重组腺病毒Ad-PML(NLS-),经4轮扩增后,检测其滴度。采用RT-PCR法和Western blot法分别检测重组腺病毒中PML(NLS-)基因mRNA的转录水平和蛋白的表达水平;将重组腺病毒感染K562细胞,MTT法检测其对细胞增殖活力的影响。结果重组腺病毒质粒pAd-PML(NLS-)经PCR和单酶切鉴定,证明构建正确;经4轮扩增,重组腺病毒的滴度为1×1010pfu/ml;Ad-PML(NLS-)携带的PML(NLS-)能够在AD293细胞中稳定表达;与未感染组和空载病毒感染组相比,重组腺病毒Ad-PML(NLS-)感染的K562细胞的增殖活力明显增强(P<0.05)。结论成功构建了重组腺病毒Ad-PML(NLS-),其可促进K562细胞的增殖,表明PML(NLS-)可能具有促癌基因的作用。     

重组抗体高效表达的研究进展(Ⅰ)

重组抗体在哺乳动物细胞中的高效表达有赖于高效表达载体的构建、抗体基因序列的优化、抗体稳定细胞株的筛选、表达宿主细胞的改造及细胞培养工艺的优化等。本文对上述问题的研究进展进行综述。     

Notch信号通路相关分子在脊椎生长过程中的差异表达及其对软骨细胞分化的调控作用

目的探讨Notch信号通路相关分子在脊椎生长过程中的差异表达及其对软骨细胞分化的调控作用。方法采用RT-PCR检测各不同发育年龄段小鼠椎间盘Notch信号分子mRNA的表达情况,筛选调控脊椎正常生长发育的重要信号分子,并采用免疫组化法进行验证。用重组腺病毒介导Dll-1、Jag-1、骨形态发生蛋白-2(bone morphogeneticprotein-2,BMP-2)在髓核(nucleus pulposus,NP)细胞中过表达,RT-PCR检测软骨细胞特征性标志物蛋白聚糖(aggrecan,ACAN)和Ⅱ型胶原(collagen typeⅡ,COL2A1)的表达,甲苯胺蓝染色检测软骨细胞基质分泌情况。结果小鼠脊椎生长发育过程中,Notch信号分子mRNA的表达总体呈下降趋势,其中Dll-1、Dll-3、Jag-1下降趋势尤其显著;Dll-1和Jag-1广泛表达于软骨细胞胞膜,随着小鼠年龄的增大,二者在椎间盘中的表达显著下降;BMP-2过表达可促进NP细胞成软骨分化,ACAN和COL2A1表达升高,甲苯胺蓝染色增强;Dll-1、Jag-1过表达可显著抑制BMP-2诱导的成软骨分化效应。结论 Notch信号分子,尤其是Dll-1、Jag-1对正常脊椎生长及椎间盘软骨细胞的成熟分化具有明显的负性调节作用。