【摘要】 目的 應用細菌內(nèi)同源重組高效制備p53基因重組腺病毒.方法 設計引物擴增p53基因,在上下游引物分別引入Xhol和HindⅢ酶切位點,將p53基因亞克隆連接到經(jīng)相同酶切的p shuttle-cmv轉(zhuǎn)移質(zhì)粒上,轉(zhuǎn)化DH5α篩選卡那霉素抗性菌落構(gòu)建重組腺病毒轉(zhuǎn)移質(zhì)粒p shuttle-cmv-p53用Pmel酶切線性化采用兩步法進行同源重組：先將腺病毒骨架質(zhì)料pAdEasy-1轉(zhuǎn)化氯化鈣法制備的BJ5183感受態(tài)細菌,篩選得到鏈霉素和氨芐青霉素抗性的AdBJ5183轉(zhuǎn)化菌；再將線性化的p shuttle-cmv-p53轉(zhuǎn)化氯化鈣法制備的AdBJ5183感受態(tài)細菌,在細菌內(nèi)進行同源重組,挑選卡那霉素抗性菌落培養(yǎng)并提取質(zhì)粒,瓊脂糖電泳挑選大片斷的重組質(zhì)粒pAdBJ5183-P53分別用限制性內(nèi)切酶PacI和BstXI及PCR法鑒定.結(jié)果 成功構(gòu)建了p53基因重組腺病毒表達載體.結(jié)論 采用簡化的兩步氯化鈣化學轉(zhuǎn)化法可以取代電穿孔法高效構(gòu)建重組腺病毒表達載體.
【關(guān)鍵詞】 腺病毒載體；同源重組；p53基因
Construction of recombinant adenoviral vector carrying wt-p53 gene by simplified two-step homologous recombination in bacteria
ZOU Chang-yong,QUANG Jia-wu,TIAN Sheng-he,et al.Wuhan Institute of Biological Products,Wuhan 430060,China
【Abstract】 Objective To construct recombinant replication-defective human adenovirus serotype 5 vector carrying wt-p53 gene by simplified two-step homologous recombination protocol in bacteria.Methods wt-p53 gene was firstly subcloned into a transfer vector p shuttle-cmv ａnd the positive recombinant p shuttle-p53 was linearized by PmeI.Then the simplified two-step homologous recombination protocol was employed for the construction of recombinant adenoviral vector.In step one,adenoviral skeletal plasmid pAdeasy-1 was transformed into BJ5183 competent cells by calcium chloride chemical methed to obtain pAdBJ5183;and in step two,linearized psh-p53 plasmid was transformed into pAdBJ5183.The possible positive adenoviral plasmids were selected by agarose gel electrophoresis ａnd indentified by PacI as well as BxtXI cleaved pattern.Results Replication-defective recombinatant adenovirus containing human p53 gene was successfully constructed.Conclusion The results indicated that the calcium chloride chemically simplified two-step homologous recombination protocol can replace the co-transformation by electroperforation ａnd may have broaed utility in system that involve homologous recombination in bacteria.
【Key words】 adenoviral vector;homologous reombination;p53 gene
復制缺陷型腺病毒可以高效介導基因的體內(nèi)外轉(zhuǎn)移；感染細胞范圍廣,不但可以感染分裂期細胞,也可感染靜止期細胞；感染細胞時其DNA不整合到宿主細胞染色體中,安全可靠.因此成為基因治療中常用的載體.重組腺病毒載體的構(gòu)建方法有真核細胞內(nèi)質(zhì)粒重組法 酵母人工染色體克隆系統(tǒng) Cre/loxp定點重組系統(tǒng) 末端蛋白-DNA復合物共轉(zhuǎn)染法等［１～３］.以上方法牽涉到的步驟和環(huán)節(jié)較多 工作量大 實驗周期長.1998年He等［4］建立了細菌內(nèi)質(zhì)粒共轉(zhuǎn)化同源重組方法,該法簡便快速 實驗周期短,但是共轉(zhuǎn)化需要電穿孔儀,且同源重組成功率不到20%.筆者在實際工作中對該法進行了改進,采用簡化的兩步氯化鈣化學轉(zhuǎn)化法代替電穿孔共轉(zhuǎn)化法構(gòu)建了p53重組腺病毒表達載體.
1 材料與方法
1.1 實驗材料 pAdEasy-1 pshuttle-cmv質(zhì)粒,BJ5183
DH5α大腸桿菌,PmeI酶購自Qbiogene公司；攜帶p53基因的質(zhì)粒由馬延高教授惠贈；PacI酶購自New England公司；PCR Premix DL2000Marker購自TaKaPa公司；λDNA·HindⅢMarker購自Promega公司；其他分子生物學工具酶購自MBI公司.
1.2 重組轉(zhuǎn)移質(zhì)粒的構(gòu)建 設計擴增p53基因的上下游引物分別為：
p53-up:5 atg tct cga gat gga gga gcc gca gtc aga 3 （30bp）,
p53-down:5 aag taa gct ttc agt ctg agt cag gcc ctt 3 （30bp）,在上下游引物分別引入XhoI和HindⅢ酶切位點,由上海生工合成.采用上述引物以攜帶p53基因的質(zhì)粒為模板進行PCR擴增p53基因片斷.PCR參數(shù)為：94℃變性5min;94℃30s 56℃30s 72℃50s,共30個循環(huán)；72℃延伸10min.用XhoI和HindⅢ酶切PCR產(chǎn)物,瓊脂糖凝膠電泳回收,連接到同樣經(jīng)XhoI和HindⅢ酶切回收的pshuttle-cmv轉(zhuǎn)移質(zhì)粒中；轉(zhuǎn)化DH5α感受態(tài)菌,卡那霉素抗性平板篩選.
1.3 重組轉(zhuǎn)移質(zhì)粒的鑒定 挑選卡那霉素抗性平板篩選菌落培養(yǎng),堿裂解法提取質(zhì)粒,用XhoI和HindⅢ酶切鑒定,并以能酶切出與目的基因片段大小相符的質(zhì)粒為模板,用上述引物進行PCR鑒定.得到的重組轉(zhuǎn)移質(zhì)粒命名為psh-p53.再用上述引物由上海生工公司測定psh-p53中目的基因片段序列.
1.4 兩步法細菌內(nèi)同源重組產(chǎn)生重組腺病毒質(zhì)粒
1.4.1 step1:用氯化鈣法制備BJ5183感受態(tài)細菌 將腺病毒骨架質(zhì)粒pAdEasy-1轉(zhuǎn)化BJ5183感受態(tài)細菌,接種在鏈霉素和氨芐青霉素抗性平板篩選,挑取菌落培養(yǎng)并抽提質(zhì)粒,以pAdEasy-1為對照,用BstXI酶切鑒定,得到的陽性菌命名為AdBJ5183菌.
1.4.2 step2:同源重組 用氯化鈣制備AdBJ5183感受態(tài)細菌；將序列正確的重組轉(zhuǎn)移質(zhì)粒psh-p53用PmeI酶切線性化,不需滅活PmeI酶,也不需回收,直接取大約30ng酶切線性化質(zhì)粒轉(zhuǎn)化AdBJ5183感受態(tài)細菌,卡那霉素抗性平板篩選,挑取菌落培養(yǎng)并抽提質(zhì)粒,0.8%瓊脂糖電泳初篩分子量大于30kbp的質(zhì)粒,以pAdEasy-1為對照,分別用PacI BstXI酶切鑒定,對酶切圖譜符合的質(zhì)粒采用前述擴增目的的基因片段的引物進行PCR鑒定.得到的陽性菌命名為AdBJ5183-p53菌.相應的陽性重組腺病毒質(zhì)粒為pAdBJ5183-p53.再將AdBJ5183P53轉(zhuǎn)化DH5α得到pAdp53.
2 結(jié)果
2.1 重組轉(zhuǎn)移質(zhì)粒psh-P53的構(gòu)建及鑒定
（1）重組轉(zhuǎn)移質(zhì)粒psh-P53經(jīng)XhoI和HindⅢ雙酶切,1%瓊脂糖凝膠電泳觀察,出現(xiàn)一條約7.4kg的轉(zhuǎn)移質(zhì)粒帶和一條約1.2kb的p53目的基因帶,見圖1.
（2）以能酶切出與目的基因片段大小相符的質(zhì)粒為模板,用擴增目的基因的引物進行PCR鑒定.可以得到約1.2kb大小的片斷,見圖2.
圖1 重組轉(zhuǎn)移質(zhì)粒的酶切鑒定
Fig1 screening recombinant transfer vecter by XhoI ａnd HindⅢ lanel:DL2000Marker lane2～4：recombinant transfer vecter/XhoI ａnd HindⅢ lane5～6：transfer vector/XhoI ａnd HindⅢ as control
圖2 重組轉(zhuǎn)移質(zhì)粒的PCR鑒定
Fiv2 screening recombinant transfer vecter by PCR lane1:negative control lane2:positive control lane3:recombinant transfer vecter lane4:DL2000 marker
（3）測序結(jié)果經(jīng)Vector NTI軟件分析正確.
2.2 重組腺病毒表達質(zhì)粒的構(gòu)建及鑒定
（1）將腺病毒骨架質(zhì)粒轉(zhuǎn)化BJ5183感受態(tài)菌,抽提鏈霉素和氨芐青霉素雙抗性的質(zhì)粒,對照骨架質(zhì)粒pAdEasy-1,用BstXI酶切鑒定.pAdEasy-1質(zhì)粒有6個BstXI酶切位點,可以產(chǎn)生6個片斷.電泳結(jié)果顯示,AdBJ5183質(zhì)粒與pAdEasy-1圖譜一致,見圖3.
圖3 pAdBJ5183質(zhì)粒用BstXI酶切鑒定
padBJ5183 plasmids cleaved pattern by BstXI lane1:λDNA/HindⅢ Marker,lane2:pAdEasy-1,lane3:pAdEasy-1/BstXI,lane4:pAdBJ5183/BstXI
（2）線性化的重組轉(zhuǎn)移質(zhì)粒psh-p53轉(zhuǎn)化AdBJ5183感受態(tài)細菌進行同源重組后,抽提卡那霉素抗性的質(zhì)粒,0.8%脂糖電泳發(fā)現(xiàn)有2種質(zhì)粒：其一分子量約34kb,為可能的重組腺病毒質(zhì)粒；另一分子量約9kb,為重組轉(zhuǎn)移質(zhì)粒psh-p53.可以很直觀方便地初篩出可能的大片斷重組腺病毒質(zhì)粒（圖略）.
PacI酶切分子量約34kb的質(zhì)粒可出現(xiàn)一條約30～35kb的大片斷和一條4.5kb的特征性片斷見圖4.與預期結(jié)果相符.
BstXI酶切鑒定重組腺病毒質(zhì)粒 PAdEasy-1質(zhì)粒有6個BstXI酶切位點,可以產(chǎn)生6個片斷,依次為11921bp 8237bp 5251bp 4254bp 2379bp 1399bp.其與線性化的psh-p53在BJ5183中發(fā)生同源重組的位置只引起片斷2的改變.本實驗采用Pshuttle-cmv為轉(zhuǎn)移質(zhì)粒,同源重組后片斷2將漂移至11～12kb左右,與片斷1重疊,只可以觀察到5條帶.與預期結(jié)果相符.結(jié)果見圖5.
圖4 重組腺病毒質(zhì)粒PAdBJ5183 p53用pacI酶切鑒定
Fig4:Identification of recombinant Adenoviral plasmids by PacI lane1:λDNA/HindⅢ Marker,lane2:PAdeasy-1/PacI,lane3-6:pAdBJ5183-p53/PacI
圖5重組腺病毒質(zhì)粒PAdBJ5183 p53用BstXI酶切鑒定
Fig5 Identification of recombinant Adenoviral plasmids by BstXI cleaven patten
lane1:λDNA/HindⅢ Marker,lane2～4：pAdBJ5183-p53/BstXI；
lane5:PAdeasy-1/BstXI；lane:PAdeasy-1
以酶切鑒定正確的重組腺病毒質(zhì)粒為模板進行PCR,可擴增出與目的基因大小相符合的片斷,見圖6.
圖6 重組腺病毒質(zhì)粒的PCR鑒定
Fig6 PCR amplified p53 gene of recombinant Adeneviral plasmids lane1:DL2000 marker；lane2-6:pAdBJ5183;lane7:psh-p53 positive control;lane8:negative control
3 討論
傳統(tǒng)的構(gòu)建重組腺病毒的方法是在293細胞內(nèi)進行質(zhì)粒間同源重組,這些方法存在細胞類同源重組效率低 實驗周期長等缺點,給研究工作帶來了諸多不便；利用生長周期快 操作方便的大腸桿菌實現(xiàn)質(zhì)粒間同源重組產(chǎn)生腺病毒的方法具有重組效率高 病毒基因組高度一致 不需蝕斑純
化篩選純系病毒等優(yōu)點.在進行細菌內(nèi)同源重組時,最初的方法是將腺病毒骨架質(zhì)粒和線性化的重組轉(zhuǎn)移質(zhì)粒電穿孔共轉(zhuǎn)化大腸桿菌BJ5183,這種方法需要電穿孔儀,而且重組效率相對較低；Zeng等［5］采用兩步法進行了改進,先用電穿孔法將骨架質(zhì)粒PAdEasy-1轉(zhuǎn)化電穿孔感受態(tài)大腸桿菌BJ5183,得到BJ5183AdEasy,再將線性化的重組轉(zhuǎn)移質(zhì)粒電穿孔法轉(zhuǎn)化電穿孔感受態(tài)大腸桿菌BJ5183AdEasy進行同源重組,由于該法是將轉(zhuǎn)移質(zhì)粒轉(zhuǎn)化到已攜帶有骨架質(zhì)粒的繁殖能力強的BJ5183菌中,避免了將轉(zhuǎn)移質(zhì)粒轉(zhuǎn)化到有缺陷或復制能力弱的細菌中,因而成功率提高到94%（16/17）,但該法仍然需要電穿孔儀,一般實驗室難以推廣.筆者將該兩步法的電穿孔部分改為傳統(tǒng)的氯化鈣化學轉(zhuǎn)化法,而且線性化的重組轉(zhuǎn)移質(zhì)粒不需經(jīng)過熱滅活PmeI酶及凝膠電泳回收,直接用于轉(zhuǎn)化,也得到了較高的重組率（6/8）.本研究正在進行p53基因重組腺病毒生物學特性的研究及對腫瘤細胞作用的研究.
作者：鄒昌勇,全家嫵,田生和,楊京生,吳季南 中華醫(yī)藥雜志
作者單位： 430060 湖北武漢,武漢生物制品研究所
【參考文獻】
1 顧建人,曹雪濤.基因治療.北京：科學出版社.2001,197.
2 Ng P,Parks RJ,Cummings DT,et al.A high-efficiency Cre/loxP-based system for constrction of adenoviral vectors.Hum Gene Ther,10:2667-2672.
3 Kamen A,Henry 0.Development ａnd optimization of an adenovirus production process.J Gene Med,2004,6:184-192.
4 He TC,Zhou S,L.T.da Costa,et al.Asimplified system for generating recombinant adenovirus.Proc Natl Acad Sci USA,1998,95（5）:2509-2514.
5 Zeng M,Smith SK,Siegel F,et al.AdEasy system made easier by selecting the viral backbone plasmid preceding homologous recombinanation.Bio Techniques,2001,13（2）:260-262.
