转铁蛋白受体单链抗体与链亲和素融合基因的克隆及其在大肠杆菌中的可溶性表达

目的:转铁蛋白受体特异性富含于血脑屏障和肿瘤细胞的表面,是当前中枢神经系统疾病和肿瘤治疗中定向转运的重要靶标。拟获得在大肠杆菌中能高效可溶表达的转铁蛋白受体单链抗体与链亲和素(SA)的重组融合蛋白。方法:根据GenBank数据库报道的SA的核苷酸序列分段合成基因,连接后经PCR获得完整的基因片段,插入pGEM-T载体中测序。将序列正确的SA基因与大鼠转铁蛋白受体单链抗体基因ox26-scFv分别插入原核表达载体pTIG-Trx中,构建重组表达克隆pTIG-Trx/scFv-SA,并在大肠杆菌中诱导表达。ELISA检测融合蛋白的生物学活性。结果:对pGEM-T/SA克隆的测序结果显示,合成的SA基因与文献报道相符。重组融合蛋白在大肠杆菌中获得了可溶性表达,约占菌体上清总蛋白量的30%;ELISA结果表明该融合蛋白具备与转铁蛋白受体和生物素的结合的双重活性。结论:有活性的重组融合蛋白的获得为构建一个通用性的以转铁蛋白受体介导的血脑屏障和肿瘤转运靶向载体打下了基础。     

大鼠转铁蛋白受体单链抗体和芋螺毒素SO3的融合蛋白在大肠杆菌中的表达

人工合成了芋螺毒素SO3的基因片段,通过链延伸方法获得了SO3的全长基因。在此基础上,将SO3基因插入到含抗大鼠转铁蛋白受体的单链抗体基因的pTIG-Trx表达载体中,构建含抗大鼠转铁蛋白受体单链抗体和SO3融合基因的重组表达载体,转化大肠杆菌BL21(DE3)和Origmia(DE3)／DsbC并得到了表达,该融合蛋白主要以包涵体形式存在。为进一步研究芋螺毒素SO3跨血脑屏障转运和治疗作用奠定了基础。     

抗转铁蛋白受体单链抗体的可溶性表达及其对脑的靶向性研究

分段合成大鼠抗转铁蛋白受体单链抗体基因(ox26-scFv),经三轮PCR拼接成794bp的完整片段。测序后构建pTIG-Trx/ox26-scFv表达载体,在大肠杆菌BL21(DE3)中获得了可溶性表达,经Ni-NTA金属鏊合层析柱纯化,在29 kD处可见单一条带。大鼠GH3细胞免疫组化显示,该单链抗体能识别并与转铁蛋白受体结合。将单链抗体尾静脉注射大鼠,于鼠脑组织切片上可见阳性染色,尤其是脑血管处着色明显,说明该单链抗体对脑血管具有较好的靶向作用,并在受体的介导下通过了血脑屏障。     

抗转铁蛋白受体单链抗体的表达、纯化及活性测定

根据转铁蛋白受体和转铁蛋白特异性结合的性质,利用亲和纯化的方法从胎盘中提取转铁蛋白受体。以转铁蛋白受体为抗原包被免疫管,从全合成人源噬菌体单链抗体库中筛选其抗体。对筛选到的抗体进行特异性鉴定后,将抗体基因插入表达载体pET22b( ),转化大肠杆菌BL21(DE3),IPTG诱导后获得可溶性表达。HeLa细胞的免疫组化结果显示,表达的抗体可以与转铁蛋白受体结合。表达产物经Ni-NTA金属螯和层析柱纯化、脱咪唑后,从尾静脉注射小鼠。1h后,去除血液及毛细血管的干扰,在脑实质中检测到了抗体的存在,说明该抗体可以通过血脑屏障。     

脑药物转运载体——抗转铁蛋白受体单链抗体的克隆表达及鉴定

为构建特异性的脑药物转运载体 ,分段合成了抗大鼠转铁蛋白受体的单链抗体基因 (Ox2 6 scfv) .经重叠PCR拼接成完整片段 ,克隆入pUC19载体中 ,测序正确后克隆到大肠杆菌表达载体pET 15b E .tag上 .IPTG诱导 ,表达产物分子量为 2 9kD ,约占菌体总蛋白量的 4 0 % .包涵体经 6mol L盐酸胍变性后 ,过SephacrylS 30 0HR分子筛柱复性蛋白 .免疫酶染色实验表明 ,该单链抗体能与转铁蛋白受体特异性结合 ,为建立以转铁蛋白受体为介导的血脑屏障转运载体打下了基础     

转铁蛋白受体及其在药物运输中的作用

血脑屏障的存在阻止了中枢神经系统疾病许多潜在治疗药物的通过.近年来主要利用脑毛细血管内皮细胞膜中的转运蛋白,如转铁蛋白受体、胰岛素受体等,将外源药物与这些受体的特异性抗体相连,通过受体介导的内吞作用将药物转运到脑组织中.转铁蛋白受体在抗癌药物定向运输及恶性肿瘤细胞基因治疗中的研究已经处于临床阶段.     

人转铁蛋白受体及其特异性抗体的制备

目的:从胎盘中提取转铁蛋白受体并获得抗转铁蛋白受体的抗体。方法:人新鲜胎盘组织被破碎后,用去污剂TritonX-100裂解细胞膜,释放膜蛋白。利用膜蛋白中的转铁蛋白受体能与铁-转铁蛋白复合物特异性结合的特性对其进行亲和纯化。对纯化得到的目的蛋白,经脱盐后进行ELISA及肽质量图谱分析,证明为所需的转铁蛋白受体后,以其包被免疫管,从全合成人源噬菌体抗体库中筛选抗体。结果:从人源噬菌体抗体库中筛选到5个能够与转铁蛋白受体特异性结合的噬菌体单链抗体。结论:以人源转铁蛋白受体为抗体,可从全人源噬菌体抗体库中筛选到其特异性的抗体。     

脑源性神经营养因子受体trkB在NIH 3T3细胞上的表达

构建了克隆有大鼠脑源性神经营养因子（BDNF）受体trkB全长基因的真核表达载体pcDNA3.1( )-rat trkB.用脂质体介导法将重组载体转入小鼠NIH3T3细胞,在mRNA和蛋白质水平检测到了trkB基因在用G418筛选到的抗性NIH 3T3细胞中的表达,表达的trkB蛋白定位于细胞膜上。BDNF能够剂量依赖性地促进NIH 3T3－trkB细胞的增殖,说明表达的trkB是有功能的。该表达trkB和NIH3T3细胞为研究BDNF的生理功能、活性测定和从噬菌体展示肽库中筛选BDNF肽提供了一个简便的细胞模型。     

脑内的铁,转铁蛋白及转铁蛋白受体

脑铁异常增高可能参与脑神经变性疾病的发生发展。这一发现使得脑铁代谢成为近年广为关注和研究较为广泛的领域。本文综述了这一领域某些方面的目前认识。包括：（１）脑铁分布及功能;（２）铁转铁蛋白及转铁蛋白受体在脑内的合成与分布;（３）脑铁摄取和运输。此外,对铁与某些金属离子,转的蛋白和转铁蛋白受体与脑神经变性疾病的关系,以及转铁蛋白受体内吞在生物大分子跨血脑屏障运输中的作用也作了简要讨论。     

在豌豆植物中瞬时表达大鼠神经营养因子BDNF

[目的]克隆大鼠神经营养因子BDNF基因,构建植物表达载体,在豌豆植物中表达BDNF蛋白。[方法]采用RT-PCR法克隆大鼠脑源性神经营养因子BDNF基因,构建豌豆植物表达载体p CAPE2-BDNF,利用豌豆发芽种子真空侵染法在豌豆植物中瞬时表达BDNF蛋白,以His标签抗体进行Western Blot检测目的蛋白。[结果]获得含有鼠源性神经营养因子BDNF基因的植物表达载体p CAPE2-BDNF,His标签抗体检测到目的条带。[结论]BDNF蛋白在豌豆植物中成功表达,有助于进一步对其功能活性进行分析。     

Preservation of the beta-galactosidase activity in E. coli cells containing the recombinant pUC19 plasmid

Two BglII fragments of pJC703 cosmid were inserted into the plasmid PUC19. E. coli cells containing the recombinant PUC19/A plasmid acquired rifampicin resistance due to inserted rpoB gene but still expressed the Lac+ phenotype.     

Efficient recovery of the functional IP10-scFv fusion protein from inclusion bodies with an on-column refolding system

A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpressed in E. coli BL21(DE3). With an on-column refolding procedure based on Ni-chelating chromatography, the active fusion protein was recovered efficiently from inclusion bodies with a refolding yield of approximate 45% confirmed by spectrophotometer. The activity of refolded IP10-scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed the fusion protein retains the specific binding activity to AIF with an affinity constant of 4.48x10(-8) M as well as the chemokine function of IP-10. The overall yield of IP10-scFv with bioactivity in E. coli flask culture was more than 40 mg/L.     

日本血吸虫(中国大陆株)谷胱甘肽转移酶编码区基因的体外扩增、克隆及在原核细胞的高效表达

为表达、获取日本血吸早谷胱甘肽转移酶（ＳｊＧＳＴ）基因工程重组蛋白,以日本血吸虫（中国大陆株）ｃＤＮＡ为模板,设计、合成特定寡核苷酸引物,ＲＴ－ＰＣＲ法扩增ＧＳＴ编码基因序列,将扩增产物连接ｐＧＥＭ－Ｔ克隆载体,再亚克隆到真、原核表达质粒ｐＢＫ－ＣＭＶ中,转染大肠杆菌ＸＬ１－ｂｌｕｅ,经ＩＰＴＧ诱导后用ＳＤＳ－ＰＡＧＥ分析表达效果。结果 ＲＴ－ＰＣＲ法特异性扩增出日本血吸虫ＧＳＴ编码区基因片段,其     

Expression and purification of functional Clostridium perfringens alpha and epsilon toxins in Escherichia coli

The alpha and epsilon toxins are 2 of the 4 major lethal toxins of the pathogen Clostridium perfringens. In this study, the expression of the epsilon toxin (etx) gene of C. perfringens was optimized by replacing rare codons with high-frequency codons, and the optimized gene was synthesized using overlapping PCR. Then, the etx gene or the alpha-toxin gene (cpa) was individually inserted into the pTIG-Trx expression vector with a hexahistidine tag and a thioredoxin (Trx) to facilitate their purification and induce the expression of soluble proteins. The recombinant alpha toxin (rCPA) and epsilon toxin (rETX) were highly expressed as soluble forms in the recipient Escherichia coli BL21 strain, respectively. The rCPA and rETX were purified using Ni(2+)-chelating chromatography and size-exclusion chromatography. And the entire purification process recovered about 40% of each target protein from the starting materials. The purified target toxins formed single band at about 42kDa (rCPA) or 31kDa (rETX) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their functional activity was confirmed by bioactivity assays. We have shown that the production of large amounts of soluble and functional proteins by using the pTIG-Trx vector in E. coli is a good alternative for the production of native alpha and epsilon toxins and could also be useful for the production of other toxic proteins with soluble forms.     

基于狂犬病病毒G蛋白scFv介导的靶向shRNA制备与鉴定

【目的】探讨以狂犬病病毒G糖蛋白单链抗体介导的载体表达shRNA靶向制剂,靶向抑制狂犬病毒复制的可行性。【方法】应用PCR技术获得狂犬病毒G糖蛋白单链抗体scFv(G)和绿脓杆菌跨膜区-酵母DNA结合结构域ETA-GAL4基因,通过搭桥PCR法获得scFv(G)-ETA-GAL4(SEG)嵌合基因;克隆至原核表达载体pET28a(+),构建重组表达质粒pET28a(+)-scFv(G)-ETA-GAL4(pET28a-SEG);在大肠杆菌BL21(DE3)中经IPTG诱导表达,利用镍柱亲和层析法纯化包涵体,经复性、鉴定制得SEG蛋白;ELISA法检测表达蛋白与狂犬病毒特异结合活性;将SEG蛋白与含shRNA的质粒(pRNATU6.3-shRNA)连接制成靶向shRNA,接入100 TCID50狂犬病毒感染BHK-21细胞,35 h观察细胞中绿色荧光蛋白(GFP)表达情况;48 h用直接免疫荧光抗体试验测定复合物抑制病毒效果。【结果】克隆得到1557 bp的SEG蛋白编码基因,大肠杆菌中成功表达57 KDa的SEG蛋白,能与抗His的单克隆抗体发生特异性反应,SEG蛋白经镍柱纯化、复性后得率为2.8 mg/mL。ELISA试验证明SEG蛋白在一定浓度范围内与RV结合呈正相关。细胞试验表明GFP在细胞内得到表达;直接免疫荧光试验测定该复合物能抑制76%病毒复制。【结论】SEG蛋白能与携带shRNA的质粒结合,可运送该质粒至RV感染BHK-21细胞中,抑制狂犬病毒的复制。     

Genetically engineered brain drug delivery vectors: cloning, expression and in vivo application of an anti-transferrin receptor single chain antibody-streptavidin fusion gene and protein.

A single chain Fv antibody-streptavidin fusion protein was expressed and purified from bacterial inclusion bodies following cloning of the genes encoding the variable region of the heavy chain and light chain of the murine OX26 monoclonal antibody to the rat transferrin receptor. The latter undergoes receptor mediated transcytosis through the brain capillary endothelial wall in vivo, which makes up the blood-brain barrier (BBB); therefore, the OX26 monoclonal antibody and its single chain Fv analog may act as brain drug delivery vectors in vivo. Attachment of biotinylated drugs to the antibody vector is facilitated by production of the streptavidin fusion protein. The bi-functionality of the OX26 single chain Fv antibody-streptavidin fusion protein was retained, as the product both bound biotin and the rat transferrin receptor in vitro and in vivo, based on pharmacokinetic and brain uptake analyses in anesthetized rats. The attachment of biotin-polyethyleneglycol-fluorescein to the OX26 single chain Fv antibody-streptavidin fusion protein resulted in illumination of isolated rat brain capillaries in confocal fluorescent microscopy. In conclusion, these studies demonstrate that genetically engineered single chain Fv antibody-streptavidin fusion proteins may be used for non-invasive neurotherapeutic delivery to the brain using endogenous BBB transport systems such as the transferrin receptor.     

Hv古细菌SRP19基因的克隆、表达、重组蛋白的纯化及生物学活性的研究

目的：构建Hv古细菌SRP19蛋白的表达载体pET23d-HvSRP19并在大肠杆菌中表达后进行纯化和研究其生物学活性,为研究SRP循环的分子机制奠定基础。方法：用体外合成的重组DNA技术,先合成具有重叠碱基的10个寡核苷酸短序列,通过拼接,获得Hv SRP19基因全长DNA后,克隆到pET23d载体上。重组质粒在大肠杆菌BL21（DE3）pLysS中的大量表达产物经Q-Sepharose离子交换层析柱纯化后再用蔗糖密度梯度超速离心法分析其生物学活性。结果：正确构建了pET23d-Hv SRP19表达载体,并在大肠杆菌BL21（DE3）pLysS中获得良好的表达;成功地纯化了表达产物,纯度达95%;证明了具有SRP19蛋白的生物学活性,能够与Hv SRP RNA相互作用形成SRP19-SRP RNA的复合物。结论：纯化的Hv SRP19蛋白与Hv SRP RNA相互作用所形成的复合物,被认为是启动SRP颗粒形成和功能发挥的开始。