抗氯霉素单链抗体的高效生产

[目的]建立抗氯霉素单链抗体(scFvCAP)的大肠杆菌高效生产体系。[方法]采用重叠延伸PCR法组装scFvCAP,并通过优化5’端序列实现高效表达,采用β-环糊精人工伴侣系统实现SDS变性蛋白的复性。[结果]成功组装scFvCAP基因(Genebank ID:GU258048)。在强启动子T7驱动下,scFvCAP基因独立表达时未见显著表达,而通过同义突变所得突变基因scFvCAP mut(Genebank ID:GU258048)即可高效表达。高效表达所形成的包涵体蛋白可溶于1%SDS-8mol/L尿素溶液,经β-环糊精人工伴侣系统复性后所获scFvCAP具有与母本单克隆抗体相似的亲和力。[结论]成功建立了基于大肠杆菌基因工程系统的scFvCAP高效生产体系。     

人FoxA1基因重组慢病毒载体的构建和鉴定

【摘要】 目的 构建叉头盒蛋白A1(Forkhead-boxA1,FoxA1)的重组慢病毒表达载体,为探究其功能和作用机制奠定基础。方法〓设计基因引物,采用聚合酶链反应(PCR)扩增出FoxA1的cDNA序列,然后将其克隆至PCDH-CMV-MCS-EF1a-GFP-puro慢病毒表达载体上,经PCR、双酶切反应及DNA测序鉴定,将阳性重组FoxA1表达载体、pCMV-VSV-G 和pCMV-dR8.91包装质粒共转染到HEK-293T细胞进行病毒包装收集病毒浓缩液,然后再感染293T细胞,通过观察绿色荧光蛋白来计算病毒滴度,最后用病毒转染原代培养人皮肤成纤维细胞(HFFs),通过实时荧光定量PCR检测FoxA1 mRNA的表达水平。结果〓重组慢病毒表达载体FoxA1经PCR扩增、酶切及测序鉴定正确,并得到滴度为1×108 TU/mL的病毒液,病毒感染人皮肤成纤维细胞后表达显著增强。结论〓成功构建了人FoxA1重组慢病毒载体,并可在HFFs内高效表达,为后续FoxA1的功能和机理研究奠定了实验基础。     

Genetic Variation in the ABCB1 Gene May Lead to mRNA Level Chabge: Application to Gastric Cancer Cases

Background: One of the major mechanisms for drug resistance is associated with altered anticancer drug transport, mediated by the human-adenosine triphosphate binding cassette (ABC) transporter superfamily proteins. The overexpression of adenosine triphosphate binding cassette, sub family B, member 1 (ABCB1) by multidrug-resistant cancer cells is a serious impediment to chemotherapy. In our study we have studied the possibility that structural single-nucleotide polymorphisms (SNP) are the mechanism of ABCB1 overexpression. Materials and Methods: A total of 101 gastric cancer multidrug resistant cases and 100 controls were genotyped with sequence-specific primed PCR (SSP-PCR). Gene expression was evaluated for 70 multidrug resistant cases and 54 controls by real time PCR. The correlation between the two groups was based on secondary structures of RNA predicted by bioinformatics tool. Results: The results of genotyping showed that among 3 studied SNPs, rs28381943 and rs2032586 had significant differences between patient and control groups but there were no differences in the two groups for C3435T. The results of real time PCR showed over-expression of ABCB1 when we compared our data with each of the genotypes in average mode. Prediction of secondary structures in the existence of 2 related SNPs (rs28381943 and rs2032586) showed that the amount of ΔG for original mRNA is higher than the amount of ΔG for the two mentioned SNPs. Conclusions: We have observed that 2 of our studied SNPs (rs283821943 and rs2032586) may elevate the expression of ABCB1 gene, through increase in mRNA stability, while this was not the case for C3435T.     

Dlx2 over-expression: a possible mechanism for first branchial arch malformation

The first branchial arch malformation(FBAM) is a rare congenital defect associated with anomalous development of the first and second branchial arches.Cause of FBAM still remains unknown,and is thought in most cases to be multifactorial,involving both genetic and enviromental factors.Dlx2 as a member of the Dlx homeobox gene family,plays a crucial role in the development of the first branchial arch.The tissues regulated mainly by Dlx2 are coincident with the tissues mainly involved in FBAM.Dlx2 over-expression generated by electroporation transfection can disturb the migration and differentiation of cranial neural crest cells(CNCCs),which migrate to the branchial arches and in turn give rise to much of the facial skeleton and connective tissues.Furthermore,Dlx2 over-expression can be found in the first branchial arch spontaneous mutant mice.So we hypothesize that Dlx2 over-expression mutation causes FBAM due to an increase in cell-cell adhesion and inhibiting the migration of CNCC to the first branchial arch in the early stage,or migrating to an incorrect position and can’t differentiate into normal tissues.What an exact role of Dlx2 over-expression in FBAM remains to be investigated and Dlx2 over-expression transgenic mouse will be a nice model for further research in FBAM.     

切口蛋白受体3在非功能性垂体腺瘤中的表达

目的 探讨切口蛋白受体3(notch3)在非功能性垂体腺瘤发病机制中的作用. 方法 收集无锡市第二人民医院神经外科自2005年1月至2011年12月收治的16例被病理证实为非功能性垂体腺瘤的肿瘤标本,以及5例正常垂体组织(通过通过组织捐赠获得),采用逆转录-聚合酶链反应、免疫印迹法、免疫组化等方法观察notch3 mRNA及蛋白在非功能性垂体腺瘤和正常垂体组织中的表达情况. 结果 在非功能性垂体腺瘤组织中,notch3mRNA及蛋白表达均显著上调,高于正常垂体组织,差异有统计学意义(P＜0.05). 结论 Notch3蛋白的过度表达可能在非功能性垂体腺瘤的生长过程中具有重要意义.     

rAcAP5高表达工程菌株的筛选、多肽的制备、纯化及对栓塞性脑缺血的溶栓作用

犬钩虫抗凝肽5（AcAP5）是一种具有抗凝活性的多肽。本实验室发现重组AcAP5（rAcAP5）具有TAFIa抑制活性,并在体内、体外实验中均具有溶栓活性。本研究拟筛选rAcAP5高表达菌株,并用大鼠栓塞性MCAO模型来评价rAcAP5的溶栓作用。根据大肠杆菌偏爱密码子对编码AcAP5的目的基因进行了优化用具有不同特点的6种表达质粒和11种大肠杆菌菌株构建了66种重组菌株,从中选出rAcAP5可溶性表达量最高的菌株采用阴离子交换色谱和阳离子交换色谱来纯化目的蛋白用高效液相色谱法测定纯化过的rAcAP5的纯度通过飞行质谱测定所表达的rAcAP5的分子量用大鼠栓塞性大脑中动脉阻塞（MCAO）模型评价溶栓作用。rAcAP5表达量最高的菌株是C2566H/pTYB1-rAcAP5,经过两步离子交换层析纯化之后,纯度达到90%,纯蛋白产量为28 mg/L。激光多普勒血流仪测定rCBF评价溶栓效果,给生理盐水（溶媒）后rCBF无明显变化,而给予rAcAP5（50–200μg/kg,i.v.）后,rCBF显著增加,剂量有依赖性,表明rAcAP5具有明显的溶栓作用。综上所述,我们筛选到rAcAP5的高表达菌株,表达、纯化并鉴定了该多肽,发现它在栓塞性MCAO模型上有明显的溶栓作用,提示它有可能作为溶栓剂或辅助溶栓剂应用。     

Enhancing production of ergosterol in Pichia pastoris GS115 by over-expression of 3-hydroxy-3-methylglutaryl CoA reductase from Glycyrrhiza uralensis

The rate-limiting enzyme in the mevalonic acid (MVA) pathway which can lead to triterpenoid saponin glycyrrhizic acid (GA) is 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR). In order to reveal the effect of copy number variation in the HMGR gene on the MVA pathway, the HMGR gene from Glycyrrhiza uralensis Fisch. (GuHMGR) was cloned and over-expressed in Pichia pastoris GS115. Six recombinant P. pastoris strains containing different copy numbers of the GuHMGR gene were obtained and the content of ergosterol was analyzed by HPLC. The results showed that all the recombinant P. pastoris strains contained more ergosterol than the negative control and the strains with 8 and 44 copies contained significantly more ergosterol than the other strains. However, as the copy number increased, the content of ergosterol showed an increasing–decreasing–increasing pattern. This study provides a rationale for increasing the content of GA through over-expressing the GuHMGR gene in cultivars of G. uralensis.KEY WORDS: Glycyrrhiza uralensis Fisch., 3-Hydroxy-3-methylglutaryl-CoA reductase gene, Over-expression, Pichia pastoris, Copy number variationAbbreviations: BMGY, buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; CNV, copy number variation; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; LOD, limit of detection; LLOQ, lower limit of quantitation; MD, minimal dextrose medium; MM, minimal medium; MVA, mevalonic acid; PCR, polymerase chain reaction; RSD, relative standard deviation; YPD, yeast peptone dextrose medium     

紫外线照射对P53基因表达的影响

目的 探讨紫外线照射对抑癌基因Ｐ５３表达的影响。方法 对Ｐ５３基因功能正常的舌癌细胞株为靶细胞,用不同剂量的紫外线照射或相同剂量紫外线照射不同时间后,采用免疫沉淀法检测Ｐ５３基因表达水平的变化。结果 紫外线照射可诱导Ｐ５３基因过度表达。Ｐ５３基因的过度表达在紫外线照射后可持续相当长一段时间。结论 外源性ＤＮＡ损伤因子可诱导Ｐ５３基因表达;Ｐ５３基因过度表达可能是细胞对外源性ＤＮＡ损伤因子造成细胞基     

A novel Toll like receptor with two TIR domains (HcToll-2) is involved in regulation of antimicrobial peptide gene expression of Hyriopsis cumingii

Animal Toll-like receptors (TLRs) are involved in innate immunity. Toll proteins are generally transmembrane proteins. In this study, an atypical Toll-like receptor (HcToll-2) was identified from the triangle-shell pearl mussel Hyriopsis cumingii, which belongs to phylum Mollusca. Unlike the typical Toll like receptors with extracellular leucine-rich repeats (LRRs), transmembrane, and intracellular Toll/interleukin-1 receptor (TIR) domains, HcToll-2 has two homologous TIR domains located at the C-terminal (designated as HcTIR1 and HcTIR2) and lacks a transmembrane domain. Phylogenetic analysis showed that HcTIR1 was clustered with TIR of sea anemone Toll, and HcTIR2 was clustered with TIR of Drosophila Toll. HcToll-2 mRNA could be detected in the hepatopancreas and was upregulated after challenge with Escherichia coli and Staphylococcus aureus. Recombinant HcLRR protein with GST tag could bind to bacteria and also to LPS and PGN. Over-expression of both HcTIR1 and HcTIR2 induced drosomycin genes in Drosophila S2 cells. RNAi analysis showed that HcToll-2 was required for the expression of theromacin, which is a cysteine-rich antimicrobial peptide (AMP) gene. This research is the first report of an atypical Toll-like receptor HcToll-2 involved in antibacterial immunity through induction of AMP expression.