Construction of Eukaryotic Expressing Vector pCH503 of CH50 and Its Chemotaxis and Anti-Tumor Function by Expression in vivo in Mice

Fibronectin (FN) is a high--molecular glycopro-tein which can be produced by many types of cells 1'nvivO and participates in many physiological and patho-logic processes. FN fragments containing Cell I do-.main are chemotactic to macrophagesL'j. We haveproduced a recombinant polypeptide CH50 by the recombination of Cell 1 and Hep I domains of humanFN[zj. CH50 can activate macrophages and inhibitthe metastasis of tumor["*'J. To investigate the function of CH50 expressed in the by gene t…     

Construction and expression of eukaryotic expressing vector pCH510 of polypeptide CH50 and its chemotaxis and antitumor function byin vivo transfection

Summary  To construct an eukaryotic expressing vector that expresses CH50, a recombinant Cell I-Hep I bifunctional-domain polypeptide of human fibronectin, and to investigate the chemotaxis to immune cells and the inhibitory effect on the growth of tumor by the expression of the plasmidin vivo, the plasmid was constructed by DNA recombination. Gene transfection was performedin vitro andin vivo. The expressed product was identified by Western blot. The chemotaxis after gene transfectionin vivo was observed by histotomy and staining of muscle tissues. The inhibition of gene transfection on solid tumor was observed in mice. The results showed that plasmid pCH510 was constructed by the recombination of the 5′-terminal noncoding region and signal peptide coding region of human fibronectin cDNA and cDNA fragment coding CH50 polypeptide with a 3′-terminal noncoding region of human FN cDNA, and the insertion of the recombinated fragment into plasmid pcDNA3. 1. After transfection with plasmid pCH510, NIH3T3 cells could produce CH50 polypeptide. The transfection of plasmid pCH510 by the injection in muscle of mouse could produce the effects of chemotaxis on immune cells and the inhibition on the growth of solid tumor. It is concluded that plasmid pCH510 can express in cells andin vivo in mouse. The expression of the plasmidin vivo has a chemotactic effect on immune cells and can inhibit the growth of solid tumor. This project was supported by a grant from the National Natural Science Foundation of China (No. 39870763) and a Funding Program for New-Century Talent of the Ministry of Education of China.     

Construction of eukaryotic expressing vector pCH503 of CH50 and its chemotaxis and anti-tumor function by expressionin vivo in mice

Summary An eukaryotic expressing vector that expresses CH50, a recombinant polypeptide of human fibronectin, in mice was constructed, and its chemotactic and anti-tumor function byin vivo gene transfection was investigated. The plasmid was constructed by recombination techniques. The cDNA fragment coding CH50 polypeptide from a prokaryotic expressing vector of CH50 was ligated with 5′-terminal noncoding region and coding region of signal peptide of mouse IFN-7 cDNA at 5′ side and 3′-terminal noncoden region of human FN cDNA at 3′ side. The recombinant cDNA was inserted into plasmid pREP8. The resulted expressing plasmid was designated as pCH503. The macrophages transfected with pCH503in vivo and culturedin vitro could produce CH50. The expressed product was identified by heparin-affinity chromatography and SDS-PAGE. By counting and Giemsa-staining of coeliac cells and histotomy and staining of muscle tissue, the chemotaxis on immune cells was observed after transfection of pCH503 either in peritoneal cavity or in muscle. The inhibition of gene transfection of pCH503 on melanoma was observed in mice. The number of melanoma nodes in mice was reduced by 50%–60% after coeliac transfection with pCH503. The pCH503, an eukaryotic expressing vector of CH50, can expressin vivo in mice. The transfection of pCH503in vivo has the chemotaxis on immune cells and can inhibit the formation of tumor nodes, suggesting that plasmid pCH503 is potentially useful in combined treatment of tumor. This project was supported by a grant from the National Natural Science Foundation of China (No. 39870763) and Trans-Century Training Program Foundation for Talents under the Supervision of Ministry of Education of China.     

CH50多肽对IFN-γ基因转染癌细胞体内生长及免疫刺激作用的影响

探讨重组FN多肽CH50对IFN-γ基因转染癌细胞体内生长及免疫刺激作用的影响。将小鼠IFN-γ基因转染黑色素瘤B16/Fl细胞,测定其表达产物与CH50协同刺激巨噬细胞产生NO的作用、转染细胞接种小鼠并注射CH50时对脾细胞的免疫刺激作用及瘤细胞的体内生长特性。结果表明,转染细胞表达产物可与CH50协同作用刺激巨噬细胞产生NO。转染细胞表达IFN-γ水平较低,仍可在体内形成肿瘤,注射CH50能够抑制其形成肿瘤。基因转染细胞和CH50能够促进脾细胞对肿瘤细胞的杀伤能力。研究表明,CH50不仅可以提高肿瘤疫苗的安全性,也可以提高肿瘤疫苗刺激机体免疫系统的作用。并提示,表达CH50和IFN-γ双因子的肿瘤疫苗可为提高肿瘤疫苗治疗肿瘤的效果开辟新的途径。     

Inhibitory Effect of recombinant FN polypeptide CH50 on the metastasis and tumor growth

Immunotherapycouldremoveresidualtumorcellsbyenhancinganti-tumorfunc-tionofimmunesystemaftersurgicalre-movaloftumor.ltisatime-consumingtofullyactivateimmunece11sandformstrongcytotoxicityontumorcellsinvivo.ltisim-perativetofindawaytoactivateanti-tumoractivityofimmunecellsquick1yenoughinvivosothattheresidualtumorcellscouldbeattackedasearlyaspossible.CH5oisabifunctiona1-domainrecombinantpolypep-tidewhichcontainsCellIandHepIdo-mainsofhumanfibronectin(FN)andisabIetoexpressinE-coli[lj.Thispolypep-…     

Inhibitory Effect of Recombinant Fibronectin Polypeptide CH50 on Invasion and Metastasis of Melanoma B16 Cells

In order to investigate the inhibitory effect and mechanism of recombinant polypeptide CH50 on invasion and metastasis of melanoma B16 cells, the recombinant polypeptide CH50 was separated and purified by ion exchange chromatographic technique. The melanoma B16 cells treated with purified CH50 were cultured in vitro, the number was counted at 4, 24, 48 and 72 h and their morphological changes were observed in order to detect their adhesion and spreading abilities. In in vivo study, the melanoma B16 cells were labeled with CFSE and treated with CH50 and then they were injected into mice via mouse-tail veins. After 5 h, the lung tissues were fixed by frozen section. Accumulation and invasion abilities of B16 cells on lung tissues were observed under the fluorescent microscopy. The results showed that the morphological character of B16 cells treated with CH50 changed greatly and the number of B16 cells treated with CH50 decreased significantly (P<0.05). The adhesion and spreading abilities of B16 cells treated with CH50 were weakened obviously and the metastasis foci on lung tissues reduced. It was concluded that the recombinant polypeptide CH50 inhibited invasion and metastasis of melanoma B16 cells on tissues and could be a prospective bio-product in tumor general therapy.     

Augmentation of recombinant CH50 polypeptide on the function of macrophages of mice during chemotherapy

CH50 is a Cell I- Hep Ⅱ bifunctional-domain recombinant polypeptide of human fibronectin expressed in E. colilll. This polypeptide can inhibit theinvasion and metastasis of tumor cells 1'n the[2] andactivate the anti--tumor activity of macrophages[']. Ithas been reported that this polypeptide andchemotherapeutic agent have a synergistic inhibitoryeffect on the metastasis of tumors[4J. In this study,we further investigated the effect of CH50 on thefunction of macrophages of mice during chem…     

可溶性BTLA真核表达载体的构建及其抗肿瘤作用

目的初步研究确定体内表达可溶性B、T淋巴细胞衰减因子（BTLA）对肿瘤生长的抑制作用，探索新的肿瘤免疫治疗途径。方法采用RT—PCR及重组DNA技术构建可溶性BTLA真核表达质粒，体外细胞转染检测重组质粒的转录表达，小鼠实体瘤模型研究基因转染抑制肿瘤生长的作用。结果用RT—PCR从小鼠脾细胞中扩增出编码BTLA胞外段cDNA，克隆至真核表达载体pcDNA3．1．获得重组表达质粒pBTLA；体外转染BHK细胞可检测到重组质粒的转录表达；肌肉内注射转染质粒pBTLA可明显抑制H22小鼠肿瘤的生长。结论可溶性BTLA能够抑制肿瘤生长．提示B7x-BTLA途径参与肿瘤免疫耐受．封闭此途径有可能成为肿瘤免疫治疗的新靶点。     

双功能域重组FN多肽抑制癌细胞浸润能力的研究

在制备了两个Cell Ⅰ-Hep Ⅱ 双结构域重组FN多肽(CH50和CH56)的基础上,研究其抑制肿瘤细胞浸润能力的作用。两个多肽的结构差异是CH50中删除了Cell I和HepⅡ之间的Ⅲ-11和ED-A结构顺序。CH50(ED_(50)为30.2 nmol/L)结合细胞的能力略高于CH56(ED_(50)为45.4 nmol/L)。两种多肽均可显著抑制黑色素瘤B16/F1细胞结合层粘素的能力,抑制作用相同。在体内肿瘤浸润抑制试验中,两种多肽均可显著抑制癌细胞浸润能力,使肺转移结节数降低80％左右。结果提示:Ⅲ-11和ED-A结构顺序对Cell Ⅰ-Hep Ⅱ 双结构域多肽结合细胞的能力有一定的影响,但删除Ⅲ-11和ED-A不是重组多肽抑制肿瘤转移的决定因素,Cell I和Hep Ⅱ 这两个结构域单独连接在一起是其抑制肿瘤细胞转移的结构基础。     

Augmentation of Recombinant Fibronectin Polypeptide CH50 on the Antitumor Function of Macrophages

Fibronectin(FN)isamulti-domainglycoproteinwithcomplicatedregulatoryfunctionsllj.TheapplicationofFNtothetherapyofdiseaseshasbeentriedsince1983,buthampered'becauseofthecomplexityofitsfunctionslll.Inrecentyears,theresearchonFNwasconcentratedonthefunctionsofitsdomainfragments.IthasbeenprovedthattheFNfragmentcontainingCellIdomaincouldenhancethefunctionofmonocyte--macrophage['--'J,suggestingthatdomainfragmentofFNcouldbeveryusefulinthetherapyofdiseases.WehaverecombinedcDNAfragmentsofCellIdoma…     

Construction of Expressing Plasmids of Recombinant FN Polypeptides with Bifunctional－domain and the Characterization of the P

ＣｏｎｓｔｒｕｃｔｉｏｎｏｆＥｘｐｒｅｓｓｉｎｇＰｌａｓｍｉｄｓｏｆＲｅｃｏｍｂｉｎａｎｔＦＮＰｏｌｙｐｅｐｔｉｄｅｓｗｉｔｈＢｉｆｕｎｃｔｉｏｎａｌ－ｄｏｍａｉｎａｎｄｔｈｅＣｈａｒａｃｔｅｒｉｚａｔｉｏｎｏｆｔｈｅＰｒｏｄｕｃｔｓＥｘｐｒｅｓｓｅ...     

Effect of polypeptide CH50 on macrophage activationin vivo and antitumor function

Surgery,chemotherapy and radiotherapy aremajor methods used in tumor therapy.After the re-moval of large tumor tissue by surgery,the mainproblem to be faced with is the inhibition and elimi-nation of the residual tumor cells in vivo. Ifmacrophages can be effectively activated,the residualtumor cells could be inhibited and eliminated as earlyas possible,the conditions favorable to the induce-mentof specificimmune response againsttumorcouldalso be created. Recombinant polypeptide CH50 wasprepar…     

Effect of Fragment Asp1961-Glu1978 in Fibronectin on the Expression of Triple-domain Polypeptide in E. coli

Thebifunctional-domainrecombinantpolypeptldeoffibronectin(FN)ispotentiallyusefulintumortherapy.Thispolypeptideyieldedtheinhibltoryeffectontumormetastasisatseveralstepsoftumorcellsinvadingothertissuesll].ThestrongerfunctionwasobtainedifCell--ndomainwasconnectedtotheC--terminalofCellI--HepIpolypeptide['].Buttheexpressinglevelofthetriple--domainpolypeptideinE.coltwasverylow[ZJ.Wereportedthepreparationofbifunctional--domainrecombinantpolypeptideofFN,whichisProl239Ser1515ofFNlinkedwithAla16…     

神经内分泌多肽7B2对T淋巴细胞的影响

观察神经内分泌多肽7B2对T淋巴细胞增殖和趋化功能的影响，当其浓度在250pg／ml和500pg／ml时，可抑制10份正常人外周血的PHA淋转率，其平均抑制率分别为26．97％、61．04％。采用微量玻片T淋巴细胞趋化试验证实．当7B2浓度在3ng／ml和5ng／ml时可抑制以白细胞介素2（IL-2）作为起化剂的6份外周血经PHA刺激的T淋巴细胞趋化率，其抑制率分别为32.78％和91．78％。表明神经内分泌多肽7B2参与T淋巴细胞的调控。     

人DC-STAMP真核表达载体的构建及鉴定

目的构建和鉴定人DC-STAMP基因的真核表达载体,并分析在293T细胞中的表达情况。方法通过反转录-聚合酶链式反应(RT-PCR)技术从人破骨细胞体外扩增DC-STAMP的cDNA片段,连接入真核表达载体pEGFP-N1-FLAG后转入293T细胞中,Western blot进行表达鉴定。结果成功扩增出人DC-STAMP全长,构建了重组质粒pEGFP-DC-STAMP-FLAG,并在其转染的293T细胞检测到融合蛋白的表达。结论成功构建了DC-STAMP融合基因表达载体pEGFP-DC-STAMP-FLAG。     

Comparative Study on the Inhibitory Effect of RecombinantFN Polypeptide CH50 and CH56 on the Metastasis ofMelanoma Cells

Intactmoleculesoffibronectinhavenoinhibitoryeffectonthemetastasisoftumorcells.RecombinantCellIorHepI(singledomain)polypeptidesoffibronectin,expressedinE.colt,arenotabletoinhibitthemetastasesoftumornomattertheywereusedaloneorasamixture.ButtheinhibitoryeffectcanbeachievedwhenCellIandHepIdomainsoffibronectinwerepreparedasarecombinantbifunctional--domainpolypeptideexpressedinE.coliLlj.ItisnotclearuntilnowthatrecombinantCellI-Hep1polypeptideinhibitsthemetastasisoftumorcellsbecauseotherdomains…     

MBP-1分子真核表达载体的构建及其在食管癌细胞株Eca109中的高表达

目的:初步探讨MBP-1的分子功能.方法:通过PCR扩增获得MBP-1基因编码序列,将其定向插入质粒pcDNA3.1(+)构建重组质粒pcDNA3.1-mbp-1,重组质粒转染体外培养的食管癌Eca109细胞株,Western-blotting检测MBP-1在食管癌细胞中的表达.结果:筛选出分子量较大的重组质粒,酶切分...     

小鼠血管抑素基因的克隆及真核表达载体构建

目的 :克隆小鼠血管抑素 (Angiostatin)cDNA ,构建其真核表达载体pCMV -Angiostatin重组质粒 ,为进一步研究其抗肿瘤作用奠定基础。方法 :根据Genebank中小鼠血管抑素基因序列 ,用RT -PCR方法从鼠肝脏中扩增出AngiostatincDNA ,连接 pMD18T载体测序 ,经测序证实后 ,通过中间载体 pKS ,构建pCMV -Angiostatin重组质粒。结果 :测序表明扩增的AngiostatincDNA序列与报道基本一致 ,AngiostatincDNA正确插入表达载体。 结论 :成功地克隆小鼠Angiostatin基因并完成其真核表达载体的构建     

小鼠CD160胞外段真核表达载体的构建和表达

目的 构建小鼠CD160胞外段的真核表达载体,并稳定转染CHO细胞进行真核表达.方法 从C57BL/6小鼠脾脏中提取总RNA,经RT-PCR扩增CD160胞外段,然后将其克隆到真核表达载体pcDNA3.1和pEGFP-N1两种质粒中,构建重组质粒psCD160和pEGFP-sCD160.重组质粒经双酶切鉴定及测序正确后,用脂质体转染CHO细胞,并用RT-PCR和Western blot检测CD160胞外段的表达,流式细胞术检测重组psCD160与其配体的结合能力.结果 获得长度约为520 bp的小鼠CD160胞外段基因,经测序证实其序列正确.用脂质体将含有CD160胞外段基因的重组质粒载体转染CHO细胞后.RT-PCR和Western blot分析证实转染的CHO细胞可表达重组的psCD160基因,并且表达的可溶性CD160可与其配体结合.结论 成功构建小鼠CD160胞外段基因的真核表达载体,并转染CHO细胞后表达出相应蛋白,为进一步研究CD160胞外段的功能效应奠定实验基础.     

PcDNA3.1-rhGM-CSF重组质粒的构建及鉴定

目的:构建PcDNA3.1-rhGM-CSF真核细胞表达载体。方法:采用PCR、T-A克隆及基因定向克隆技术,将rhGM-CSF基因插入PcDNA3.1(+)空载体,构建了PcDNA3.1-rhGM-CSF真核表达载体。经限制性内切酶酶切和DNA测序进行鉴定。结果:双酶切及DNA测序证实PcDNA3.1-rhGM-CSF真核细胞表达载体构建成功。结论:成功构建PcDNA3.1-rhGM-CSF真核细胞表达载体。