[發(fā)明專利]人FHL1C真核表達(dá)載體的構(gòu)建及其應(yīng)用

有效

• 申請(qǐng)?zhí)枺?/td>: 201010557788.8
• 申請(qǐng)日: 2010-11-24
• 公開(kāi)（公告）號(hào): CN102061310A
• 公開(kāi)（公告）日: 2011-05-18
• 發(fā)明（設(shè)計(jì)）人: 秦鴻雁;梁英民;付偉;韓驊
• 申請(qǐng)（專利權(quán)）人: 中國(guó)人民解放軍第四軍醫(yī)大學(xué)
• 主分類號(hào): C12N15/85
• 分類號(hào): C12N15/85;A61K48/00;A61P35/02
• 代理公司: 西安吉盛專利代理有限責(zé)任公司 61108
• 代理人: 鮑燕平

1.人FHL1C真核表達(dá)載體的構(gòu)建方法

將FHL1C基因插入T載體，以EcoRI和SalI酶切后回收基因片段，以EcoRI和SalI酶切載體pEGFP-C1后回收載體片段，將這兩片段經(jīng)DNA連接酶進(jìn)行連接反應(yīng)，連接產(chǎn)物轉(zhuǎn)化感受態(tài)細(xì)胞XL10，挑取單克隆培養(yǎng)過(guò)夜，提取質(zhì)粒，經(jīng)EcoRI和SacII酶切鑒定，對(duì)獲得預(yù)期大小的插入片段的載體進(jìn)行測(cè)序，測(cè)序正確的載體命名為pEGFP-C1-FHL1C。

2.過(guò)表達(dá)人FHL1C在急性T淋巴細(xì)胞白血病治療中的應(yīng)用。

3.根據(jù)權(quán)利要求1所述的人FHL1C真核表達(dá)載體的應(yīng)用：其特征是：離心收集Jurkat細(xì)胞并進(jìn)行細(xì)胞計(jì)數(shù)(1×10⁶)，取針對(duì)Jurkat細(xì)胞進(jìn)行轉(zhuǎn)染的轉(zhuǎn)染液100ul重懸細(xì)胞，加入pEGFP-C1-FHL1C質(zhì)粒混合后移入Nucleofector^TM核酸轉(zhuǎn)染儀配套的電轉(zhuǎn)杯；然后將電轉(zhuǎn)杯放入轉(zhuǎn)染儀，選擇特定轉(zhuǎn)染程序X-01進(jìn)行轉(zhuǎn)染。轉(zhuǎn)染完畢，取出小杯用500μl培養(yǎng)基沖洗后移入12孔板，補(bǔ)充2mlComplete?RPMI培養(yǎng)基進(jìn)行培養(yǎng)；

構(gòu)建的人FHL1C真核表達(dá)載體pEGFP-C1-FHL1C，通過(guò)核酸轉(zhuǎn)染方式導(dǎo)入Jurkat細(xì)胞，過(guò)表達(dá)FHL1C通過(guò)誘導(dǎo)Jurkat細(xì)胞凋亡而殺死Jurkat細(xì)胞，故過(guò)表達(dá)FHL1C具有抗腫瘤的用途。

4.根據(jù)權(quán)利要求1所述的人FHL1C真核表達(dá)載體的構(gòu)建方法：No1：構(gòu)建pEGFPC1-FHL1C核酸序列

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No2：構(gòu)建pEGFPC1-FHL1C氨基酸序列

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