CONSTRUCTION AND IDENTIFY OF PROKARYOTIC VECTOR OF TIMP-3
CONSTRUCTION AND IDENTIFY OF PROKARYOTIC VECTOR OF TIMP-3
复制标题
TIMP-3原核载体的构建及鉴定
DOI：
10.1136/heartjnl-2012-302920a.286
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发表时间：
2012-10
期刊：
影响因子：
5.7
通讯作者：
gao xin
中科院分区：
文献类型：
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作者：
duan lianxin;sun xin;tian dan;gao xin
目的构建Timp-3（金属蛋白酶组织抑制剂3）原核载体并对其进行鉴定。方法提取人胎盘组织总RNA，RT-PCR扩增基因，将片段化基因连接T载体，进行克隆。并通过 BamHI 和 XbaI 切割 Timp-3-pMD18-T。琼脂糖凝胶电泳观察结果。经BamHI和XbaI酶切后一定酶切成功，与CNKI中的Timp-3测序相同，此质粒保留到大肠杆菌DH5α中。结果琼脂糖凝胶电泳鉴定：Timp-3-pMD18-T经酶切后得到Timp-3基因片段。经生工生物科技（上海）有限公司测序，结果与基因库Timp-3基因相同，批准Timp-3原核载体构建及鉴定权。方法从人胎盘组织中提取总RNA。首先，我们准备好无RNA水，并使用无RNA玻璃瓶，将DEPC放入超纯水中至终浓度0.1%，搅拌过夜后，高压灭菌。其次，取0.1 g组织，该组织为人胎盘粉末，与500 ml RNAAiso plus混合后，匀浆，室温静置5 min，加入1/5 RNAAiso Plus体积的氯仿，摇动、搅拌15s。室温静置5 min后，4℃12 000 r/min离心15 min。将上清液倒入新的附加管中。加入与上清液相同的等体积异丙醇，室温静置10 min，4℃12 000 r/min离心10 min。弃上清，加入75%乙醇1 ml沉淀，吹扫沉淀，12000 r/min，4℃离心15 min。弃上清，保留沉淀，在空气中蒸发沉淀5～10 min，加入DEPC处理水20 μl，即得总RNA。然后进行琼脂糖凝胶电泳鉴定，并将总RNA保存至-80℃。备用引物中表达装置正义和反义酶切位点，它们是BamHI、XbaI。正义：5'-CGGGATCCATCCCCTTGGCTCGGGCTCATC-3'；反义：5'-GCTCTAGAGGGGTCTGTGGCATTGATGATGATGC-3'。该引物由上海生工有限公司合成。 RT-PCR Mgcl2 2 μl，10×RT Buffer 1 μl，dNTP 1 μl，RNase Inhibitor 0.25 μl，AMV 0.5 μl，oligodt 0.5 μl，总RNA 4 μl，我们使用DEPC处理水至10 μl，杜克混合。 42℃ 30分钟，99℃ 5分钟，组成第一条cDNA链。在反转录结果模板上加入5×PCR Buffer 10 μl、Taq酶0.25 μl、sense 0.5 μl、untisence 0.5 μl，用DEPC处理水至50 μl扩增PCR：94℃升温2 min，94℃30 s， 55°C，30 s，72°C 90 s，放大30 圈，在 72°C 下拉伸 7 分钟。然后通过琼脂糖凝胶电泳鉴定PCR产物，并观察结果。 BamHI和XbaI切割 取上述DNA 1 μl，BamHI1 μl和Ifor 1 μl，10×KBuffer 1 μl，高压双蒸水13 μl，37℃放置3 h。然后，琼脂糖凝胶DNA片段纯化，通过琼脂糖凝胶电泳鉴定，并观察结果。 TA 连接和转化 pMDTM18-T Vector 1 0.5 μl，InserDNA3 4.5 μl，solutionI5 μl，总体积为 10 μl。将上述液体放入200μl感受态细胞中，并将所有液体置于冰上30分钟。混匀1790 μl LB培养基，37℃振荡培养1 h，4000 rpm离心10 s，弃上清，再用200 μl LB培养基悬浮。将菌液铺于含有氨苄青霉素的LB琼脂平板上，铺满各处，室温放置20～30 min，37℃库氏培养12 h～16 h。筛选菌落 挑取转化后的单克隆菌落，并使菌体大量增殖，提取质粒。质粒经BamHI和XbaI酶切，BamHI和XbaI酶切后产物琼脂糖凝胶电泳鉴定，上海生工有限公司测序，继续培养。结果挑取总RNA 用trizo试剂挑取总RNA，琼脂糖凝胶电泳结果如图1。RT-PCR扩增产物评价。人胎盘组织总RNA经RT-PCR扩增，琼脂糖凝胶电泳可见633 bp阴影下的特异性条带，与预期片段大小相等，如图2。将Timp-3基因与pMD™18-T连接后，将其切割，如图3。通过 BamHI 和 XbaI 酶切，琼脂糖凝胶电泳可在 2692 bp 和 633 bp 阴影处看到清晰条带，如图 3。上海生工有限公司测序，如图 4。 RT-PCR 扩增产物评价 RT-PCR 扩增的人胎盘组织总 RNA，琼脂糖凝胶可见特异性条带 633 bp阴影处电泳，与预期片段大小一致，如图2。Timp-3基因与pMD™18-T连接后，进行切割，如图3。通过BamHI和XbaI切割，琼脂糖凝胶电泳，在2692 bp和633 bp阴影处可见清晰条带，如图3。由上海生工测序有限公司测序，如图如图 4 所示。 抑制MMPs活性、诱导细胞凋亡的功能位点分离出的N端结构域、C端结构域具有结合ECM的功能。6,7正是基于这些功能，本实验构建了Timp-3的原核载体，旨在通过抑制MMPs活性、诱导细胞凋亡的功能位点，研究动脉粥样硬化的功能，治疗肿瘤奠定基础。 定位的N端结构域、C端结构域具有结合ECM的功能。6,7结论Timp-3具有天然的抑制MMPs的作用，Timp-3可以通过抑制MMPs的活性8来抑制肿瘤的侵袭、增殖、血管的发育和转移。除稳定TNF-α受体、Fas外，促进fas诱导细胞凋亡。4,5 Timp-3可以通过抑制MMPs活性而诱导细胞凋亡的功能位点分离出N端结构域、C端结构域具有结合ECM的功能。6,7正是基于这些功能，本实验构建了Timp-3的原核载体，我们的研究 旨在研究粥样斑块的功能，为肿瘤的治疗奠定基础。
Objectives constructing prokaryotic vector of Timp-3 (tissue inhibitor of metalloproteinase-3) and identify it. Methods The total RNA was extracted from human placenta tissues, amplify the gene by RT-PCR and the fragmental gene connect T vector, then clong. And cut Timp-3-pMD18-T by BamHI and XbaI. Agrose gel electrophoresis observe the result. After cut by BamHI and XbaI, enzyme must succeed, sequencing the same like Timp-3 in CNKI, This plasmid was reserved into E. coli DH5α. Results By agrose gel electrophoresis identification: We can get Timp-3 gene fragment after cut Timp-3-pMD18-T. By Sangon Biotech (shanghai) Co. Ltd. Sequence, the result same to Gene Bank's Timp-3 gene, and approve right for construction and identify of prokaryotic vector of Timp-3. Methods The total RNA was extracted from human placenta tissues. The first, we have prepare RNA-free water, and use RNA-free glass bottle, put DEPC in ultra pure water to 0.1% final concentration, after agitate pass the night, autoclave sterilisation. Secondly, We have collect 0.1 g tissue that the tissue is pulv human placenta, after mix 500 ml of RNAAiso plus, homogenate, quiescence at room temperature for 5 min, add to chloroform of 1/5 RNAAiso Plus volume, joggle and durchmischung for 15s. After quiescence at room temperature for 5 min, 12 000 r/min centrifugal 15 min at 4°C. Make supernatant into the new append off tube. Add equal volume isopropanol the same as supernatant, quiescence at room temperature for 10 min, 12 000 r/min centrifugal 10 min at 4°C. Abandon supernatant, put 75% alcohol into sedimentation with 1 ml to purge sedimentation, 12000 r/min centrifugal 15 min at 4°C. Abandon supernatant and keep the sedimentation, and evaporate sedimentation for 5–10 min in the air, then add disposal water by DEPC with 20 µl, we have get total RNA. Then by agrose gel electrophoresis identification, and keep total RNA to −80°C. in reserve primer express Device sense and antisense restriction enzyme cutting site, they are BamHI, XbaI. Sense: 5′-CGGGATCCATCCCCTTGGCTCGGGCTCATC-3′; antisense: 5′-GCTCTAGAGGGGTCTGTGGCATTGATGATGC-3′. This primer synthetise by Sangon Ltd. of Shanghai. RT-PCR Mgcl2 2 µl, 10×RT Buffer 1 µl, dNTP 1 µl, RNase Inhibitor 0.25 µl, AMV 0.5 µl, oligodt 0.5 µl, total RNA 4 µl, we use disposal water by DEPC to 10 µl, durchmischung. 42°C 30 min, 99°C 5 min, to compose the first chain of cDNA. At the moulding board of reverse transcription outcome, add 5×PCR Buffer 10 µl, Taq enzyme 0.25 µl, sense 0.5 µl, untisence 0.5 µl, we use disposal water by DEPC to 50 µl amplification PCR: rise in temperature for 2 min at 94°C, 94°C 30 s, 55°C, 30 s, 72°C 90 s, amplification 30 circles, stretch for 7 min at 72°C. Then by agrose gel electrophoresis identification to PCR production, and observed result. Cut by BamHIand XbaI Get above DNA 1 µl, BamHI1 µl andIfor 1 µl, 10×KBuffer 1 µl, high pressure double distilled water 13 µl, put it for 3 h at 37°C. Then, agarose gel DNA fragment purification by agrose gel electrophoresis identification, and observed result. TA linkage and conversion pMDTM18-T Vector 1 0.5 µl, InserDNA3 4.5 µl, solutionI5 µl, total volume is 10 µl. Put above liquid in competent cell of 200 µl, and put all liquid on ice for 30 min. Mix LB culture media of 1790 µl, oscillating culture for 1 h at 37°C, 4000 rpm centrifugal 10 s, abandon supernatant, by LB culture media of 200 µl suspend again. Put bacterial liquid shop in LB agar plate contain ampicillin, spread on everywhere, put it 20–30 min at room temperature, and culture it for 12 h to 16 h in couveuse at 37°C. Screening bacterial colony Picking monoclone bacterial colony after conversion, and make the germ a lot of proliferation, then we have extract plasmid. The plasmid is cut by BamHIand XbaI, agrose gel electrophoresis identify product after cut by BamHIand XbaI, sequencing by Sangon Ltd. of Shanghai, continue to have the culture. Results Picking total RNA Picking total RNA with trizo reagents, the result by agrose gel electrophoresis is figure 1. Appraisal of amplificative production by RT-PCR. Total RNA of human placenta tissues amplificative through RT-PCR, we can see specificity band by agrose gel electrophoresis in the shadow of 633 bp, equal to expect segment magnitude as figure 2. After link Timp-3 gene and pMD™18-T, cut this, as Fig.3. Through BamHIand XbaI were cut, by agrose gel electrophoresis, we can see clear band in the shadow of 2692 bp and 633 bp as figure 3. By Sangon Ltd. of shanghai sequencing, as in figure 4. Appraisal of amplificative production by RT-PCR Total RNA of human placenta tissues amplificative through RT-PCR, we can see specificity band by agrose gel electrophoresis in the shadow of 633 bp, equal to expect segment magnitude as figure 2. After link Timp-3 gene and pMD™18-T, cut this, as figure 3. Through BamHIand XbaI were cut, by agrose gel electrophoresis, we can see clear band in the shadow of 2692 bp and 633 bp as figure 3. By Sangon Ltd. of shanghai sequencing, as in figure 4. through inhibit MMPs' activity and induced cells apoptosis' functional locus isolated N-extremity structural domain, C-extremity structural domain have function to combine ECM.6,7 Because of these function, constructing prokaryotic vector of Timp-3 this experiment, Our studies aim to research function of atheroma, treat tumours to lay a foundation, through inhibit MMPs' activity and induced cells apoptosis' functional locus islocated N-extremity structural domain, C-extremity structural domain have function to combine ECM.6,7 Conclusions Timp-3 is natural inhibit MMPs, Timp-3 can through inhibit MMPs' activity,8 to stifle the tumours invasion, proliferation, blood vessel of development and metastasis. In addition to stable TNF-αreceptor, Fas, and promote the fas induced cells apoptosis.4,5 Timp-3 can through inhibit MMPs' activity and induced cells apoptosis' functional locus isolated N-extremity structural domain, C-extremity structural domain have function to combine ECM.6,7 Because of these function, constructing prokaryotic vector of Timp-3 this experiment, Our studies aim to research function of atheroma, treat tumors to lay a foundation.