大鼠高亲和力二羧酸转运蛋白的克隆表达及细胞内定位

目的：克隆大鼠高亲和力钠依赖二羧酸转运蛋白（SDCT2）的全长cDNA,并明确其在肾小管上皮细胞内的定位表达情况。方法：从大鼠肾组织中提取总RNA,用带有8肽FLAG标签的PCR引物通过RT-PCR技术扩增出SDCT2全长基因并测序,将其插入真核表达载体中构建SDCT2真核表达载体,然后将它们转染到肾小管上皮细胞LLC-PKl中表达,并用激光共聚焦显微镜观察该蛋白的亚细胞定位情况。结果：扩增出2kb的SDCT2全长基因,Westernblot表明肋CT2基因在LLC-PKl细胞中得到了正确的表达;共聚焦显微镜分析显示正常SDCT2蛋白主要定位于细胞膜上,与生物信息学的预测结果一致;为了比较不同连接温度对重组DNA连接效率的影响,观察了3种连接温度下的转化效率,结果显示温度循环法的连接效率明显比其他2种常规连接温度要高。结论：高亲和力钠依赖二羧酸转运蛋白全长基因被成功克隆,其主要表达于肾小管上皮细胞膜上。     

巴西固氮螺菌Yu62的EGFP标记及其在小麦体内的定殖研究

以质粒pEGFP-C1为模板,采用PCR方法特异性扩增增强型绿色荧光蛋白(EGFP)基因全长序列,将其与原核表达载体pVK-100连接,构建成重组载体pVK-EGFP.利用电转化法将重组载体导入巴西固氮螺菌Yu62中,得到EGFP标记菌株.用EGFP标记菌接种小麦'小偃107'种子,室内限菌条件下培养10 d后,用荧光显微镜观测标记菌在小麦体内的定殖规律并观察接菌植株的田间生长状况.结果显示,巴西固氮螺菌Yu62能定殖于小麦根毛区、茎组织的细胞间隙等部位,而且接菌小麦'小偃107'植株在根系发育、株高、分蘖数等方面比对照有较明显的优势.研究表明,巴西固氮螺菌Yu62能够定殖于小麦根茎内,并具有促进植物生长的作用.     

Expression of Streptococcus mutans wall-associated protein A gene in Chinese hamster ovary cells: prospect for a dental caries DNA vaccine.

The Streptococcus mutans strain GS-5 wall-associated protein A (Wap-A) is a precursor to the extracellular antigen A (AgA), a recognized candidate dental caries vaccine. The full-length wapA gene (wapA-E) and a C-terminal truncated version (wapA-G) encoding the AgA were cloned into the mammalian expression vector pcDNA 3.1/V5/His-TOPO. The resulting constructs were propagated in the Escherichia coli Top10. To investigate the expression of the S. mutans genes in mammalian cells, the above constructs were used to transfect Chinese hamster ovary (CHO) cells in the presence of the cationic lipid pfx-8. Transient expression of the wapA-E and wapA-G genes was observed at 24 h post-transfection, as shown by Western immunoblot analysis using a rabbit antiserum to S. mutans cell wall. Immunochemical staining of the transfected CHO cells showed expression of WapA mainly in the cells and budding vesicles, whereas AgA was found mainly in the transfected cells and extracellular medium. The expression of S. mutans proteins in CHO cells, in either vesicles or soluble form, suggested an antibody response to the above DNA constructs. Work is under way to test the efficacy of these as DNA vaccines against S. mutans.     

A novel and simple method for construction of recombinant adenoviruses

Recombinant adenoviruses have been widely used for various applications, including protein expression and gene therapy. We herein report a new and simple cloning approach to an efficient and robust construction of recombinant adenoviral genomes based on the mating-assisted genetically integrated cloning (MAGIC) strategy. The production of recombinant adenovirus serotype 5-based vectors was greatly facilitated by the use of the MAGIC procedure and the development of the Adeasy™ adenoviral vector system. The recombinant adenoviral plasmid can be generated by a direct and seamless substitution, which replaces the stuff fragment in a full-length adenoviral genome with the gene of interest in a small plasmid in Escherichia coli. Recombinant adenoviral plasmids can be rapidly constructed in vivo by using the new method, without manipulations of the large adenoviral genome. In contrast to other traditional systems, it reduces the need for multiple in vitro manipulations, such as endonuclease cleavage, ligation and transformation, thus achieving a higher efficiency with negligible background. This strategy has been proven to be suitable for constructing an adenoviral cDNA expression library. In summary, the new method is highly efficient, technically less demanding and less labor-intensive for constructing recombinant adenoviruses, which will be beneficial for functional genomic and proteomic researches in mammalian cells.     

血管内皮生长因子全长抗体在毕赤酵母中的表达与活性鉴定

目的:构建血管内皮生长因子(VEGF)全长抗体表达载体pPICZαA-VH-CH-VL-CL,评价其在毕赤酵母中的表达产物与抗原的结合特性,及其抑制细胞增殖的活性。方法:利用基因合成分别获得VEGF抗体CL和CH序列,分别构建pPICZαA-CH和pPICZαA-CL重组质粒,再利用同尾酶特性构建双启动子表达盒的重组pPICZαA-CH-CL载体,用Westen印迹对其进行表达鉴定后将VH和VL序列插入该载体,获得VEGF的全长抗体表达载体pPICZαA-VH-CH-VL-CL;通过膜筛和ELISA进行菌株筛选,并对VEGF抗体表达阳性菌株进行小量表达和纯化,采用CCK-8法对其抑制人脐静脉内皮细胞(HUVEC)增殖的活性进行初步评价。结果:获得表达轻、重链的VEGF抗体表达载体,ELISA实验证明pPICZαA-VH-CH-VL-CL具有一定的VEGF抗原结合特性;体外增殖实验表明,该抗体可以以剂量依赖性抑制HUVEC增殖。结论:在毕赤酵母中表达、纯化了具有一定功能活性的VEGF全长抗体,为后续比较研究酵母糖基化改造对VEGF抗体的药效学和药代学的影响提供了基础。     

Construction of a human full-length cDNA bank

We aimed to construct a full-length cDNA bank from an entire set of human genes and to analyze the function of a protein encoded by each cDNA. To achieve this purpose, a multifunctional phagemid shuttle vector, pKAl, was constructed for preparing a high-quality cDNA library composed of full-length cDNA clones which can be sequenced and expressed in vitro and in mammalian cells without subcloning the cDNA fragment into other vectors. Using this as a vector primer, we have prepared a prototype of the bank composed of full-length cDNAs encoding 236 human proteins whose amino acid sequences are identical or similar to known proteins. Most cDNAs contain a putative cap site sequence, some of which show a pyrimidine-rich conserved sequence exhibiting polymorphism. It was confirmed that the vector permits efficient in vitro translation, expression in mammalian cells and the preparation of nested deletion mutants.     

Use of conversion adaptors to clone antigen genes in lambda gt11

A strategy has been devised and tested to employ EcoRI conversion adaptors for cloning 5' cohesive-ended restriction fragments into the unique EcoRI site of the lambda gt11 expression vector. Five lambda gt11 chromosomal libraries were constructed with Rickettsia tsutsugamushi genomic DNA digested with restriction enzymes generating five different 5' cohesive ends. Recombinant phage yields as high as 10(7) plaque forming units were achieved without amplification of the five libraries. Sequences encoding epitopes of all eight R. tsutsugamushi polypeptide antigens, previously identified by Western blot analysis, were obtained in the five lambda gt11 expression libraries. Recombinant antigen expression was dependent on lambda gt11 lac promoter induction in 39% of the recombinants assayed. This method significantly improves the efficiency of genomic lambda gt11 library construction by eliminating blunt-ended ligation and simplifying the removal of unligated EcoRI-ended oligonucleotides.     

Human anti-EGFL7 recombinant full-length antibodies selected from a mammalian cell-based antibody display library

Epidermal growth factor-like domain 7 (EGFL7) has been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. The advent of antibody display technology (phage, bacteria, and yeast) led to an enormous revival in the use of antibodies as diagnostic and therapeutic tools for fighting cancer. However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. We describe here the isolation of an EGFL7-specific antibody from a mammalian cell-based full-length antibody display library generated from peripheral blood mononuclear cells of patients with hepatocellular carcinoma. Using a novel vector, contained glycosylphosphatidylinositol anchor and restriction enzyme sites NheI and ClaI, antibody libraries are displayed as whole IgG molecules on the cell surface and screened for specific antigen binding by a combination of magnetic beads and measured by cell ELISA. Anti-EGFL7 antibody was successfully isolated from the library. The mammalian cell-based full-length antibody display library is a great potential application for rapid identification and cloning of human mAbs of targeting hepatocellular carcinoma.     

包装信号可以被Cre重组酶切除的辅助性腺病毒的构建及鉴定

目的:为了探索构建第三代复制依赖性腺病毒载体系统的有效实验方法,利用细菌内重组原理,分别构建了第三代腺病毒生产体系中的辅助性腺病毒和表达Cre重组酶的真核表达载体,并分析了Cre切除辅助病毒包装信号的有效性。方法:通过PCR及酶处理法依次将2个同向的loxP位点及绿色荧光蛋白表达盒克隆至pShuttle穿梭载体质粒,按照AdEasy系统的标准实验方法产生和扩增辅助病毒;同时构建pcDNA3.1(+)-cre真核表达载体,并用该载体经脂质体法转染HEK293细胞,通过PCR和流式细胞术分析转染细胞内表达的Cre重组酶对随后感染的辅助病毒的包装信号的切除作用。结果:构建的辅助性腺病毒能够在HEK293细胞内扩增,其包装信号可以由细胞内表达的Cre重组酶有效切除。结论:构建了辅助性腺病毒和pcDNA3.1(+)-cre真核表达载体,为第三代腺病毒的生产奠定了实验基础。     

A high-throughput platform for population reformatting and mammalian expression of phage display libraries to enable functional screening as full-length IgG

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.     

Development of a novel mammalian cell surface antibody display platform

Antibody display systems have been successfully applied to screen, select and characterize antibody fragments. These systems typically use prokaryotic organisms such as phage and bacteria or lower eukaryotic organisms, such as yeast. These organisms possess either no or different post-translational modification functions from mammalian cells and prefer to display small antibody fragments instead of full-length IgGs. We report here a novel mammalian cell-based antibody display platform that displays full-length functional antibodies on the surface of mammalian cells. Through recombinase-mediated DNA integration, each host cell contains one copy of the gene of interest in the genome. Utilizing a hot-spot integration site, the expression levels of the gene of interest are high and comparable between clones, ensuring a high signal to noise ratio. Coupled with fluorescence-activated cell sorting (FACS) technology, our platform is high throughput and can distinguish antibodies with very high antigen binding affinities directly on the cell surface. Single-round FACS can enrich high affinity antibodies by more than 500-fold. Antibodies with significantly improved neutralizing activity have been identified from a randomly mutagenized library, demonstrating the power of this platform in screening and selecting antibody therapeutics.Key words: antibody display, mammalian display, antibody library, vector, antibody screen, affinity maturation     

Secretion of rat serum albumin by COS cells transfected with a spliced cDNA gene. A system to study protein sorting

Certain structural features of secreted proteins may function as "sorting signals" to direct the various steps required in the secretory pathway. In order to identify and study the function of these signals we have cloned a complete cDNA gene encoding rat serum albumin (RSA) and expressed this gene in COS-1 cells via an SV40-plasmid shuttle vector. The gene was constructed by splicing together a segment of genomic DNA and three cDNA fragments excised from recombinant plasmids. DNA endonuclease digestion and ligation at restriction sites common to overlapping regions of these four RSA DNA fragments assured the maintenance of the translation reading frame during the construction of this gene. COS-1 cells transfected with the recombinant vector containing the full-length RSA gene (pSV2rsa) synthesize and secrete RSA immunoreactive material into the culture medium. This mammalian expression system provides a means to study the signals and processes involved in intracellular transport of secreted proteins.     

Humanization and Characterization of an Anti-Ricin Neutralization Monoclonal Antibody

Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9) variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with K_D of 1.63 nM, comparable to its parental murine D9 (2.55 nM). In a mouse model, intraperitoneal (i.p.) administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.     

Isolation and characterization of bovine and mouse terminal deoxynucleotidyltransferase cDNAs expressible in mammalian cells.

We have isolated nearly full-length cDNA clones of terminal deoxynucleotidyltransferase (TdT) from calf thymus and mouse lymphoma cDNA libraries. The libraries were constructed using the pcD vector system which permits the expression of cDNA inserts in mammalian cells. The bovine TdT cDNA clone contains an open reading frame coding for 520 amino acids, Mr 59,678. The mouse TdT cDNA clone contains an open reading frame of 1,587 bp, whose translated cDNA encodes a 60,004 dalton protein. The mouse TdT cDNA clone contains 60 bp in the 3' end region of the coding sequence not found in the bovine TdT cDNA sequence, otherwise, the clones share about 80% homology. A possible nuclear-localization-sequence (Pro-Arg-Lys-Lys-Arg-Pro-Arg) was conserved in the N-terminal region in the mouse and bovine cDNA clones. Bovine and mouse cDNAs transfected into COS7 monkey fibroblasts directed the synthesis of enzymatically active protein of Mr 60,000 which was detected immunologically using polyclonal rabbit antibody against bovine TdT. Bovine TdT expressed in COS7 cells by nearly full-length cDNA clone was localized in the nucleus and the translational product of pOK103 lacking the nuclear-localization-sequence was localized in the cytoplasm.     

Purification and characterisation of a soluble N-terminal fragment of the breast cancer susceptibility protein BRCA1

The BRCA1 gene encodes a large multidomain protein of 1863 residues, mutations in which lead to breast cancer. Studies to elucidate the mechanisms by which BRCA1 prevents tumour formation have been restricted by the size of the protein. Unable to purify large amounts of the full-length protein, we have identified a fragment of BRCA1, amino acid residues 230-534, that when cloned into the expression vector pET 22b and expressed in Escherichia coli is found predominantly in the soluble portion of the cell lysate. The resulting protein was purified to homogeneity and studies reveal that BRCA1 230-534 binds specifically to four-way junction DNA when compared to duplex and single-stranded DNA.     

A rapid DNA assembling strategy mediated by direct full-length polymerase chain reaction

An efficient DNA assembling strategy was developed here modified from Class-IIS endonuclease mediated DNA splicing by directed ligation (SDL). Benefited from the full-length PCR directly using ligation products as template, this strategy required less effort and less time to obtain the assembled full-length DNA. The advantages of this strategy made it a rapid and easy-to-perform gene splicing and multiple site-directed mutagenesis approach especially practicable when more fragments need to be assembled at the same time.     

α1A-肾上腺素受体与骨形成蛋白-1片段在人胚肾293细胞中的结合

用酵母双杂交方法发现骨形成蛋白-1(bone morphogenetic protein-1,BMP-1)的片段可以与α1A-肾上腺素受体(adrenergic receptor,AR)的细胞内游离C末端结合。进一步探讨了二者在哺乳动物表达系统人胚肾293细胞(human embryonic cell 293,HEK293)中的相互作用。采用PCR方法构建含BMP-1片段eDNA的真核表达质粒PCP3HA,将其与含全长人α1A-AR eDNA的质粒PDT-α1A分别或共同转染HEK293细胞,用免疫印迹法检测到α1A-AR和BMP-1在HEK293细胞有相应的蛋白表达。用酶联免疫吸附实验对免疫印迹鉴定过的细胞裂解液进行检测,观察到空白对照组,单独转染PDTα1A-和单独转染PCP3HA的细胞,OD490值分别为0.034±0.027、0.042±0.019、0.030±0.0096,三者之间无显著性差异。共转染PDT-α1A和PCP3HA的细胞,OD490值为0.57±0.12,较其它三组具有显著性差异(均P<0.001)。免疫共沉淀结果显示,单独转染PDT-α1A或PCP3HA的细胞,免疫沉淀产物中均不能检测到BMP-1片段,只有共转染PDT-α1A和PCP3HA的细胞,在其免疫沉淀产物中能检测到BMP-1片段。ELISA和免疫沉淀结果均表明α1A-AR与BMP-1的片段在HEK293细胞中存在蛋白水平的相互作用。     

Expression of a heterodimeric Fab antibody protein in one cloning step.

A new method is presented for creating antibody expression libraries in Escherichia coli. Rather than perform two cloning steps to express the heavy and light chains of the antigen binding domain, we have used a fusion-PCR method to link the coding regions for heavy and light chain sequences in a single DNA molecule prior to vector ligation. This greatly simplifies the construction of antibody expression clones.