Specific killing effect of diphtheria toxin A fragment under control of DF3 promotor on human breast cancer cells

Objective: To study the effects of recombinant expression vector containing human breast cancer DF3 promotor and diphtheria toxin A fragment on human breast cancer cells. Methods: Constructing recombinant expression vector PGL3-DF3-DTA and transfecting it into human breast cancer cells of DF3 positive and negative. By means of RT-PCR to measure the expression of DTA in human breast cancer cells. MTT color-imetry was used to examine the effect of PGL3-DF3-DTA on growth of human breast cancer cells. By experiment on nude mice to observe the killing effect of PGL3-DF3-DTA on human breast cancer cells. Results: Recombinant expression vector PGL3-DF3-DTA was highly expressed in human breast cancer cell line of DF3 positive, and it could kill the human breast cancer cells not only in vitro but also in vivo. Conclusion: Recombinant expression vector PGL3-DF3-DTA could produce specific killing effect on human breast cancer cell line of DF3 positive.

Plasmid instability when the hsp60 gene promoter is used to express the protective non-toxic fragment B of the diphtheria toxin in recombinant BCG

The genetic modification of the live attenuated Mycobacterium bovis BCG to deliver a protective Corynebacterium diphtheriae antigen in vivo could be a safer and less costly alternative to the new and more expensive DTP vaccines available today, in particular to third world-countries. The stability of expression of heterologous antigens in BCG, however, is a major challenge to the use of live recombinant bacteria in vaccine development and appears to be dependent to a certain extent, on a genetic compatibility between the expression cassette within the plasmid construct and the mycobacterium host. In the quest for the best recombinant BCG transformant to express the dtb gene of C. diphtheriae we generated two new rBCG strains by transforming the Moreau substrain of BCG with the mycobacterial expression vectors pUS973 and pUS977, each one carrying a different promoter to drive the expression of the target antigen. After transformation recombinant BCG clones were selected on Middlebrook 7H10 kanamycin Agar plates, expanded in Middlebrook 7H9 kanamycin Broth and analyzed by agarose gel electrophoresis and immunoblotting. rBCGs transformed with the construct carrying the weak PAN promoter from M. paratuberculosis stably expressed the dtb gene. Conversely, rBCGs transformed with the construct carrying the strong mycobacterium hsp60 promoter were unstable and consequently unfit for the expression of the C. diphtheriae gene.

含EWS-FLI1结合序列的DTA载体对报告基因表达抑制的研究

目的 研究含尤文家族肿瘤EWS FLI1结合序列的DTA载体对报告基因表达的抑制作用。方法 构建含有EWS FLI1结合序列和白喉毒素A链基因的表达载体 pS2 DTA。共同转染不同剂量梯度的 pS2 DTA和pS2到尤文肉瘤细胞和对照细胞 ,检测荧光强度。结果 每增加一个 pS2 DTA剂量梯度 ,在尤文肉瘤细胞中都可以检测到虫荧光素酶的表达显著降低 ,而在对照细胞中这种作用却不明显 ;在同一个pS2 DTA剂量梯度下 ,虫荧光素酶在尤文肉瘤细胞的表达都显著高于非尤文肉瘤细胞。结论 pS2 DTA转染可以抑制尤文肉瘤细胞内其它基因表达。

PGL3-DF3-DTA在DF3阳性人乳腺癌细胞移植瘤中的表达和作用

目的探讨含人乳癌DF3/MUC1启动子转录调控序列的DTA重组表达载体PGL3-DF3-DTA对乳腺癌DF3阳性癌细胞的选择性表达和杀伤作用。方法将乳腺癌的两种细胞株MCF-7和MDA-MB-231分别接种于裸鼠背侧皮下,7 d后开始用重组的质粒进行活体内瘤注射治疗。肿瘤接种后第29天脱颈椎处死裸鼠,测量移植瘤的体积和质量;并采用逆转录-聚合酶链反应法(RT-PCR)检测裸鼠移植瘤内DTA的表达情况。结果重组表达载体PGL3-DF3-DTA在MCF-7细胞株移植瘤中表达,明显抑制肿瘤生长。结论重组表达载体PGL3-DF3-DTA对乳腺癌DF3阳性的癌细胞起到了选择性表达和杀伤作用。

DF3调控下的白喉毒素A片段在体内特异性杀伤人乳腺癌细胞的研究

目的研究新构建的含人乳腺癌DF3启动子和白喉毒素A片段的重组表达载体PGL3-DF3-DTA在体内对人乳腺癌细胞的特异性杀伤作用。方法4周龄裸鼠36只,随机分为实验组、空载体组、空白对照组、阴性对照组4组,每组9只。将DF3阳性和阴性的人乳腺癌细胞MCF-7和MDA-MB-231分别接种于相应的裸鼠皮下。待成瘤后,将重组表达载体PGL3-DF3-DTA和PGL3-DF3多次、多位点注射入相应裸鼠移植瘤内。连续测量瘤体大小,计算瘤体重量。在不同时间分批处死动物,用光镜进行形态学观察、用TUNEL法检测肿瘤细胞的凋亡、用免疫组化技术检测肿瘤内淋巴管和血管的生成情况。结果成功的建立了人乳腺癌裸鼠动物模型,经重组表达载体PGL3-DF3-DTA作用后的DF3阳性人乳腺癌细胞出现明显的凋亡现象,LYVE-1、F8基因表达水平降低。结论重组表达载体PGL3-DF3-DTA能在体内对DF3阳性的乳腺癌细胞产生特异性杀伤作用。

新构建的含DF3启动子和白喉毒素A片段的重组表达载体对人乳腺癌细胞的特异性杀伤作用

目的研究新构建的含人乳腺癌DF3启动子和白喉毒素A片段的重组表达载体PGL3-DF3-DTA对人乳腺癌细胞的影响。方法构建重组表达载体PGL3-DF3-DTA,并用其转染DF3阳性和阴性的乳腺癌细胞MCF-7和MDA-MB-231。通过MTT法测定PGL3-DF3-DTA在体外对乳腺癌细胞生长的影响,建立裸鼠动物模型观察PGL3-DF3-DTA在体内对乳腺癌细胞的杀伤效应。结果成功构建出重组表达载体PGL3-DF3-DTA并建立了人乳腺癌裸鼠动物模型,经重组表达载体PGL3-DF3-DTA作用后的DF3阳性人乳腺癌细胞出现明显的凋亡现象,Ki-67、bcl-2基因表达水平降低,bax基因表达水平升高。结论重组表达载体PGL3-DF3-DTA能对DF3阳性的乳腺癌细胞产生特异性杀伤作用。

白喉毒素A片段的表达纯化与单克隆抗体制备

白喉毒素 (Diphtheriatoxin ,DT)A片段 (DTA)是白喉毒素的酶活性区 ,也是DT类免疫毒素的关键结构域。DTA蛋白及其单克隆抗体在免疫毒素的毒性机理、检测与纯化研究等方面具有重要价值。通过在E .coli中表达了DTA ,经Q SepharoseFF和Ni2 + Sepharose两步层析纯化 ,得到纯度约为 90 %的融合蛋白。以DTA为抗原免疫BalB c小鼠 ,获得了分泌抗DTA特异单抗的杂交瘤细胞株 3B6和 3B9。单抗为IgG1亚型 ,滴度达 1∶10 6 以上 ,与DTA的结合可被抗DT马血清竞争抑制。抗DTA单抗用于免疫印迹试验 ,或制备成免疫亲和柱纯化基于DT的重组免疫毒素 ,均获得较好效果 。

Cell-specific expression of the diphtheria toxin A-chain coding sequence induces cancer cell suicide

OBJECTIVE: To test whether the diphtheria toxin A (DT-A) chain coding sequence linked to murine immunoglobulin Kappa light chain (IgKappa) promoter and enhancer have selective cytocidal effects on IgKappa producing cells. METHODS: The diphtheria toxin A gene or beta galactosidase (beta-gal) gene were linked to a murine IgKappa promoter and enhancer to construct pcDNA3IgKappaDTA or pcDNA3IgKappaLacZ plasmids. These plasmids were transfected into IgKappa producing or non-producing cells by the liposome coated DNA method. Expression of beta-gal activity and effects on cell growth of transfected cells were assessed. RESULTS: The beta-gal gene, under the control of cytomegalovirus (CMV) promoter, can express in all cell lines. Expression of beta-gal under the control of the IgKappa promoter was detected only in the IgKappa producing cell line, CA46. Expression of beta-gal was greatly suppressed when cotransfected with pcDNA3IgKappaDTA in CA46 cells.Cell growth of CA46 cells transfected with pcDNA3IgKappaDTA plasmid was significantly inhibited compared with CA46 cells transfected with pcDNA3IgKappaLacZ. CONCLUSION: Selective killing of IgKappa producing cells can be attained by introducing the diphtheria toxin A gene under the control of IgKappa promoter and enhancer.

白喉毒素DT_(386)片段的表达纯化及细胞毒性研究

基于白喉毒素 (diphtheriatoxin ,DT)的免疫毒素在临床试验中普遍发现毒性反应 ,但机制尚不清楚。据推测 ,免疫毒素的细胞毒性源自DT的酶活性区和跨膜转运区 (N端 386～ 388个氨基酸 ,DT3 86)。为考察DT3 86毒素片段与毒性反应的关系 ,将DT3 86在E .coli中进行了表达、纯化 ,并初步观察了其细胞毒性。经Q SepharoseFF离子交换层析 ,获得纯度达 95 %的DT3 86蛋白。DT3 86杀伤HL60细胞的IC50 为 2× 1 0 -6mol L ,比免疫毒素的特异杀伤毒性低 1 0 0倍以上 ;但在高浓度下 ,DT3 86可非特异性杀伤多种细胞。小鼠体内实验初步结果表明 ,给药剂量高则体内毒性反应强烈。结果提示免疫毒素的剂量限制毒性与DT3 86的非特异细胞毒性有一定相关性 。