單純皰疹病毒I型胸苷激酶基因部分缺失重組質(zhì)粒的構(gòu)建及序列分析

1、    單純皰疹病毒I型胸苷激酶基因部分缺失 重組質(zhì)粒的構(gòu)建及序列分析*        關(guān)鍵詞：?jiǎn)渭儼捳畈《拘剀占っ富蛉笔蛔冃蛄蟹治稣耗康模簶?gòu)建HSV-1型胸苷激酶基因部分缺失的重組質(zhì)粒并進(jìn)行序列測(cè)定。方法：以pUC18/TK重組質(zhì)粒DNA為模板，用PCR方法擴(kuò)增TK5端503bp基因片段，并將擴(kuò)增的片段克隆入載體pUC18中(pUC18/TK1)；用PstI和Hind雙酶切pUC18/TK，獲得TK3端383bp片段，將此酶切片段克隆入pUC18/TK1中

2、，并進(jìn)行測(cè)序。結(jié)果：構(gòu)建成重組質(zhì)粒pUC18/TK。經(jīng)酶切鑒定獲得1條約900bp的基因片段；序列測(cè)定證實(shí),HSV-1 17synTK基因缺失了第493744位251對(duì)堿基。結(jié)論：成功地構(gòu)建了部分缺失的HSV-1/TK基因重組質(zhì)粒，為研制其突變株奠定了基礎(chǔ)。中國(guó)書資料分類號(hào)：Q78R373.11Construction and seguencing recombinat plasmid of the thymidine kinase gene deletion mutation of herpes smplex virus type IZhen Rongfen, Yu Qigui, Chen

3、Wei， Fan Rong, Xue Caifang (Laboratory of Anti? infectious Diseases,the Fourth Military Medical University, Xi'an 710032) Keywords：herpes simplex virus thymidine kinase deletion mutation seguencing mutation Abstract：Aim:Constructing and seguencing recombinated plasmid of the thymidine kinase gen

4、e deletion mutation of herpes smplex virus type I (HSV-1). Methods: The 5-TK 503bp was amplified by PCR. And the TK genic DNA of HSV-1 17syn strain in the recombinated plasmid pUC18(pUC18/TK) was used as tamplate. The amplified fragment was cloned into pUC18 vector（ pUC18/TK1）. Then the pUC18/TK was

5、 cleaved with both pst I and Hind III enzymes. The 3-TK 383bp fragment was obtained and cloned into plasmid pUC18/TK1. It's sequence was analysed. Results:The recombination plasmid pUC18/TK was constructed. The 251 bp from location 493 to 744 of TK gene of HSV-1 17syn strain was deleted in the p

6、lasmid pUC18/TK . The other part of the sequence did not vary. Conclusion:The recombinated plasmid of HSV-1/TK in which part genome was delected was successfully constructed.The plasmid pUC18/TK laid a good fundation for further constructing TK deletion mutant of HSV-1. 單純皰疹病毒I型（herpes simplex virus

7、 type I,HSV-I）的胸苷激酶(thymidine kinase,TK)基因?yàn)槠湓缙诨?若發(fā)生突變則形成突變株tk()HSV-I,能在鼠三叉神經(jīng)節(jié)細(xì)胞等非分裂細(xì)胞中建立潛伏感染，但不能激活1，故對(duì)正常神經(jīng)細(xì)胞無破壞作用。但tk()HSV-I能感染分裂細(xì)胞(如神經(jīng)膠質(zhì)瘤細(xì)胞和視網(wǎng)膜瘤細(xì)胞等)，并能在這些細(xì)胞中增殖，使之溶解破壞2。體外研究表明，tk()HSV-I突變株可殺傷U87和L19等神經(jīng)膠質(zhì)瘤細(xì)胞3,4。經(jīng)皮下、腎包膜下及顱內(nèi)右前葉直接注射tk()HSV-I突變株于荷神經(jīng)膠質(zhì)瘤的裸鼠及具有免疫活性的大鼠體內(nèi)，可使腫瘤組織發(fā)生壞死，但不破壞正常組織。因此,本研究擬構(gòu)建部分缺失的HS

8、V-1/TK基因重組質(zhì)粒,以為進(jìn)一步構(gòu)建TK基因部分缺失的重組HSV-1突變株奠定基礎(chǔ)。1材料和方法1.1質(zhì)粒重組質(zhì)粒pUC18/TK(含HSV-1 17syn株TK基因的全部編碼區(qū)序列),由本室構(gòu)建5。1.2引物通過計(jì)算機(jī)輔助分析，根據(jù)HSV-1 17synTK基因序列，在其5端設(shè)計(jì)一段引物（引物1），引入EcoRI酶切位點(diǎn)；根據(jù)HSV-1 17synTK基因第468492位堿基序列，設(shè)計(jì)一段反向引物（引物2）,并引入PstI酶切位點(diǎn)。引物由美國(guó)OperonTechnologies公司合成。其序列如：引物1:For:5-CGGAATTCATGGCTTCGTACCCCTGCCATCA-3引物2

9、:Rev:5-TACTGCAGATGGCGGTCGAAGATGAGGGTGA-31.3質(zhì)粒的提取、酶切、連接及轉(zhuǎn)錄主要方法均參照分子克隆一書進(jìn)行。1.4HSV-1 17synTK基因部分序列（503bp）重組質(zhì)粒的構(gòu)建PCR反應(yīng)體系：模板為本室構(gòu)建的重組質(zhì)粒pUC18/TK600ng,引物1For和引物2Rev各1mol/L，4種dNTP各200 mol/L，3mmol/LMgCl2,5 l10×PCR緩沖液，總體積501。PCR反應(yīng)液經(jīng)100煮沸變性5min后，每管加入Taq聚合酶0.8U,以201液體石蠟油封面后，進(jìn)行PCR擴(kuò)增：即941min,601min,72延伸90s,共3

10、5個(gè)循環(huán)。然后于72延伸10min。PCR產(chǎn)物在含0.5mg/L溴化乙錠(EB)的10g/L瓊脂糖凝膠中電泳,在紫外燈下觀察結(jié)果。經(jīng)10g/L低熔點(diǎn)瓊脂糖凝膠電泳回收503bp片段。用SurePure DNA Extraction Kit(Embi Tec公司產(chǎn)品)純化回收的DNA片段。經(jīng)EcoR I和Pst I雙酶切后,克隆入經(jīng)相應(yīng)酶切后制備的pUC18載體中,轉(zhuǎn)化感受態(tài)JM109細(xì)菌。將得到的陽(yáng)性克隆，經(jīng)PCR及EcoR I和Pst I雙酶切鑒定，命名為pUC18/TK1重組質(zhì)粒。1.5HSV-1TK基因部分缺失重組質(zhì)粒的構(gòu)建用Pst I和Hind III雙酶切pUC18/TK重組質(zhì)粒，回

11、收TK3端383bp的片段；克隆入用同種酶切消化的pUC18/TK1中，轉(zhuǎn)化感受態(tài)JM109細(xì)菌，將得到的陽(yáng)性克隆，經(jīng)PCR及EcoR I和Hind III雙酶切鑒定，命名為pUC18/TK重組質(zhì)粒（1）。1pUC18/TK重組質(zhì)粒的構(gòu)建Fig 1 Construction of recombinat plasmid pUC18/TK1.6pUC18/TK序列的測(cè)定pUC18/TK重組質(zhì)粒經(jīng)用QIAGEN公司產(chǎn)品QIAamp Tip25-Kit純化后，由美國(guó)夏威夷大學(xué)生物中心Neil博士在Applied Biosystems Model 373A Version全自動(dòng)序分析儀中進(jìn)行5測(cè)序。2結(jié)

12、果2.1pUC18/TK1重組質(zhì)粒DNA的鑒定用引物1For和引物2Rev，對(duì)pUC18/TK重質(zhì)粒進(jìn)行擴(kuò)增。將擴(kuò)增產(chǎn)物克隆入pUC18載體中，經(jīng)轉(zhuǎn)化得到的陽(yáng)性克隆，用EcoR I及Pst I雙酶切和PCR鑒定，獲得預(yù)期的503bp基因片段（2）。2重組pUC18/TK1的PCR鑒定Fig 2 Identification of the recombinant plasmid pUC18/TK1 by PCR 1,2,3 and 5: Positive colonies;4 and 9: DNA molecular weight markers;6 and 7: Negative coloni

13、es 2.2pUC18/TK重組質(zhì)粒DNA的鑒定將構(gòu)建的pUC18/TK重組質(zhì)粒，用EcoR I和Hind III雙酶切消化及PCR擴(kuò)增鑒定，得到1條預(yù)期約900bp的基因片段(3)。3重組pUC18/TK質(zhì)粒的酶切鑒定Fig 3 Identification of the recombinant pUC18/TK plasmid with EcoRI and Hind III1:Negative control; 2:DNA molecular weight makers; 3,4,5,6 and 7:Positive colonies 2.3pUC18/TK重組質(zhì)粒DNA的序列測(cè)定序列測(cè)定

14、的結(jié)果顯示,pUC18/TK重組質(zhì)粒DNA缺失了HSV-117Syn株TK基因第493744位251對(duì)堿基序列，其余基因序列相同。缺失后的TK基因序列約為900bp（4）。4pUC18/TK重組質(zhì)粒的DNA序列Fig 4 Nucleotide sequence of the recombinant plasmid pUC18/TKDotted line shows deletion of nucleotide sequence 3討論TK基因是腫瘤基因治療中應(yīng)用最多、最重要的藥物敏感靶基因之一。將HSV-1TK基因克隆入腺病毒或逆轉(zhuǎn)錄病毒載體中，經(jīng)包裝細(xì)胞包裝成對(duì)快速分裂的細(xì)胞具有感染性的重組

15、病毒，將其感染腫瘤病毒細(xì)胞，使TK基因與腫瘤細(xì)胞基因組DNA整合，便可成為抗皰疹病毒核苷類藥物(如無環(huán)鳥苷和丙氧鳥苷等)的靶基因，達(dá)到殺傷腫瘤細(xì)胞的目的。將TK基因去除一小段克隆入載體中，與野生型HSV-1TK基因組DNA在活細(xì)胞內(nèi)重組后，可篩選出HSV-1TK基因部分缺失的突變株，這種突變株不能在正常神經(jīng)膠質(zhì)細(xì)胞中增殖，但能在膠質(zhì)細(xì)胞瘤細(xì)胞等快速分裂的神經(jīng)細(xì)胞中增殖并使之溶解破壞，因而可用重組的HSV-1TK基因部分缺失的突變株治療神經(jīng)膠質(zhì)瘤等腦部的惡性腫瘤。本研究采用生物工程技術(shù)，將HSV-117synTK基因的5端503bp片段與3端383bp片段連接，構(gòu)建成缺失第493744位251對(duì)

16、堿基序列的TK基因載體，為構(gòu)建TK基因部分缺失的重組HSV-1突變株，治療神經(jīng)膠質(zhì)瘤等腦部惡性腫瘤奠定了基礎(chǔ)。*?國(guó)家自然科學(xué)基金資助項(xiàng)目，NO.39570807作者簡(jiǎn)介：甄榮芬，女，52歲，副教授。西安市長(zhǎng)樂西路17號(hào),Tel(029)3374536作者單位：第四軍醫(yī)大學(xué)基礎(chǔ)部抗感染研究室，西安，710032參考文獻(xiàn)1Coen DM, Kosz-Vnenchak M, Jacobson JG, et al.Thymidine kinase-negative herpes simpex virus mutants establish lantency in mouse trigeminal b

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