WO2012163119A1 - 溶肿瘤能力增强的B型人腺病毒Ad11突变体的构建和应用 - Google Patents

溶肿瘤能力增强的B型人腺病毒Ad11突变体的构建和应用 Download PDF

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WO2012163119A1
WO2012163119A1 PCT/CN2012/071757 CN2012071757W WO2012163119A1 WO 2012163119 A1 WO2012163119 A1 WO 2012163119A1 CN 2012071757 W CN2012071757 W CN 2012071757W WO 2012163119 A1 WO2012163119 A1 WO 2012163119A1
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adll
sequence
gene
tumor
gfp
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PCT/CN2012/071757
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French (fr)
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王尧河
姜国忠
黄汉熙
曹风雨
莱蒙·尼克
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北京锤特生物科技有限公司
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Priority to JP2014513035A priority Critical patent/JP5943996B2/ja
Priority to EP12792810.9A priority patent/EP2716764B1/en
Publication of WO2012163119A1 publication Critical patent/WO2012163119A1/zh
Priority to US14/093,078 priority patent/US9315827B2/en
Priority to US15/098,342 priority patent/US9932606B2/en
Priority to US15/904,408 priority patent/US10294493B2/en

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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
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Definitions

  • the present invention relates to adenoviral vectors and their use, and in particular to a method for constructing B-type recombinant human adenovirus vectors Adl l-5EP and Adll-5ETel-GFP and applications thereof.
  • adenovirus receptor CAR
  • the intravenous adenovirus is quickly inactivated by the antibody and complement.
  • the adenovirus can be engulfed by the Kuffer cell, and the virus hexon protein and Coagulation factor X binds and then transduces hepatocytes, so excess adenovirus is likely to cause significant hepatotoxicity and liver damage.
  • Adl l is a subtype of human adenovirus type B. In terms of oncolytic treatment, Adll is clearly superior to Ad5. In addition to the CD46 receptor, Adll can also bind to other cell surface receptors X. Tuve et al reported that Adll is the only virus in the B adenovirus subgroup that binds both CD46 and X, indicating that Adl l can infect a wider range of tumor cells, thereby reducing the down-regulation of viral receptors in Ad5 applications and resulting in low infection rates. Puzzle. Another advantage of Adll over Ad5 is that the neutralizing antibodies of Adl l in the population are 10-31% lower, Ad5 is 45-90%, and there is no cross-activity in the middle.
  • Adll When Adll was intravenously injected into transgenic mice expressing CD46, no significant intrahepatic transduction and hepatotoxicity were observed. Moreover, Adll can efficiently transduce dendritic cells, so recombinant Adll can express tumor-specific antigens and enhance immune response to better use in tumor immunotherapy.
  • Ad5 used in vitro experiments with human tumor cell lines and human prostate cancer cell line DU 145 in vivo to compare the tumor-dissolving efficiencies between Ad5, Ad6, Adl and Ad35, and found that Ad5, Ad6 and Adl l have similar anti-tumor effects. And Ad35 has no anti-tumor effect. Importantly, only Ad5 produced liver toxicity. Later, a chimeric tumor-dissolving Ad5 (replacement of Ad5 cilia into B sub-aggregate adenovirus) was established, in order to enhance the anti-tumor effect by binding the chimeric oncolytic Ad5 to the CD46 receptor on the cell membrane. But with the complete subgroup B adenovirus In comparison, this method still does not overcome the neutralizing ability against the Ad5 hexon antibody.
  • CTCs circulating tumour cells
  • the level of CTCs in peripheral blood of patients with cancer can be used as a basis for monitoring and adjusting treatment and prognosis.
  • the application of immunocytokine analysis and quantitative PCR has been able to detect small amounts of CTCs in the blood.
  • the lack of specific biomarkers and high detection costs limit the popularization of these methods.
  • Adll-5EP also provides a type B recombinant human adenoviral vector for treating tumors or detecting tumor cells in the blood circulation
  • the inventors first compared the anti-tumor potential of Adll and Ad5 in human tumor cell lines in vitro, and found that only 9 of the 25 cell lines tested were sensitive to Adll. Among them, PC-3 is not sensitive to Ad5 and sensitive to Adll. Compared with Ad5, Adll significantly inhibited the subcutaneous tumor growth of PC-3 cells in vivo and increased the tumor-free rate of tumor-bearing animals. Similarly, the above experiments were repeated with MIA PaCa-2 cells that were sensitive to Ad5 and were not sensitive to Adll, and the anti-tumor ability of Adll was significantly reduced.
  • Adll receptors Despite the widespread high expression of Adll receptors in human tumor cells, wild-type Adlls are not effective in killing these tumor cells.
  • the inventors conducted extensive and in-depth research on this. Using two different methods, the inventors demonstrated that more Adlls are adsorbed on the tumor cell membrane than Ad5, and this result is independent of whether the cell is sensitive to Adll.
  • the adsorbed Adll virus particles can effectively enter the nucleus, indicating that there is a higher level of Adll in the nucleus in the early stage of viral infection than Ad5.
  • Adll El A mRNA directly affects viral replication, so down-regulation of Adll E1A mRNA in MIA PaCa-2 and LNCaP cells reduces viral replication levels and correspondingly reduces the synthesis capacity of hexon protein. This finding is consistent with the Adll originally observed. Low levels of virions are produced consistently with cytotoxicity.
  • Adll-5EP tumor-targeting adenovirus vector
  • Adll-5ETel-GFP replication-selective adenovirus
  • a method for constructing a recombinant human adenoviral vector Adll-5EP which uses a homologous recombination to replace the 365 bp gene upstream of the Ad5 E1A coding sequence, including the enhancer and promoter of Ad5 E1A, into human adenovirus type 11
  • the corresponding region of the Adll was constructed into a recombinant human recombinant adenovirus vector Adll-5EP.
  • the left arm sequence is the 392 bp segment gene of the Adll genome
  • the right arm is the Ad5 E1A enhancer and the 195-559 bp segment gene of the promoter sequence and the 568-1125 bp segment gene of the Adll ElA, in order
  • the fragments were ligated together, and the left and right arms were ligated to the cloning site on both sides of pSS-Chl to establish the shuttle vector pSS-AlA7 ; PSS-A1A7 was digested and purified by Pmel, and the Pmel digested fragment was simultaneously with pAdll at BJ5183.
  • the cells were homologously recombined and then screened on agar plates containing ampicillin and chloramphenicol; the positive clones were digested with Swal, and the anti-chloramphenicol gene expression cassette sequence was removed to generate pAdll-Ad5EP, pAdll. -Ad5EP was linearized by Notl digestion and transfected into 293 cells to produce adenovirus Adll-5EP.
  • concentrations of ampicillin and chloramphenicol are 100 mg/ml and 25 mg/ml, respectively.
  • a method for transforming a selectively soluble tumor adenovirus using the B-type recombinant human adenovirus vector Adll-5EP is one of the following methods:
  • the virus is selectively allowed to replicate in tumor cells.
  • a method for constructing a B-type recombinant human adenovirus vector Adll-5ETel-GFP comprising the following steps:
  • the left arm sequence is the first 392 bp sequence of the Adll genome
  • the right arm is the 195-378 bp gene of the Ad5 E1A enhancer
  • the -714-Obp gene of the human TERT promoter is 568- of the Adll E1A gene.
  • the 1125 bp sequence was ligated together to form a fragment, and two restriction enzyme sites Xbal and NcoI were introduced on both sides of the human TERT promoter, and the left and right arms were inserted into the corresponding sides of the pSS-Chl, respectively. Snabl and EcoRV, eventually generating pSSENTel;
  • pSSGFP Construction of pSSGFP, the left arm is the Adll genome from 27301 to lj 27837 bp DNA fragment and EGFP gene through Ncol junction product, and introduced SnaBI site at 3' of EGFP; right arm is Adll genome from 28337 to 28920 bp a DNA fragment, the left arm and the right arm are blunt-ended to the Snabl and EcoRV sites on both sides of the pSS-kna, respectively, to finally generate pSSGFP;
  • pSSENTel and pSSGFP were digested and purified by Pmel, respectively.
  • Two Pmel digested fragments were simultaneously homologously recombined with pAdll in BJ5183 cells, and then agar in three antibiotics containing ampicillin, kanamycin and chloramphenicol. Screening was performed on the plate; positive clones were digested with Swal and Sbfl, and the anti-chloramphenicol gene expression cassette sequence and kanamycin gene expression cassette sequence were removed to generate pAdll-5ETel-GFP; pAdll-5ETel-GFP was treated with Notl enzyme. After linearization, transfected into 293 cells to produce adenovirus Adll-5ETel-GFP.
  • the concentrations of ampicillin, kanamycin and chloramphenicol are 100 mg/ml, 50 ug/ml, and 25 mg/ml, respectively.
  • the Tel sequence in the pSSENTel can be replaced by a promoter of another tumor-specific gene to generate a tumor-specific oncolytic adenovirus; the GFP sequence in the pSSGFP can be replaced by another signal gene or a therapeutic gene.
  • the promoter of the Adll 18.5 gene in the pSSGFP can be replaced by a stronger tumor-specific promoter.
  • the tumor-targeting adenovirus vector Adll-5EP of the present invention is based on a wild-type Adll, and an E1A enhancer and a promoter thereof are substituted with an enhancer and a promoter of Ad5 E1A, and the vector ratio is Wild-type Adll has a stronger tumor-dissolving effect, thereby enhancing the killing ability of tumor cells.
  • the tumor-targeting adenovirus vector Adll-5EP of the present invention has tumor targeting and antitumor effects.
  • the lytic potential test showed that Adll-5EP showed better cell killing ability than Ad5 and produced stronger cytotoxicity than Adll.
  • the determination of tumor growth and tumor clearance showed that Adll-5EP can effectively reduce the growth rate of tumors, and the tumor-free rate of tumor-bearing mice is significantly better than Adll.
  • the tumor-targeted adenovirus vector Adll-5EP of the present invention can be used as a tumor-targeted genetic engineering drug for treating tumors, and will have better social and economic benefits.
  • Adll-5ETel-GFP The method for constructing the B-type recombinant human adenovirus vector Adll-5ETel-GFP of the present invention, the homologous recombination of the constructed shuttle vector pSSENTeK pSSGFP and Adll-5EP genome simultaneously produces recombinant virus Adll-5ETel-GFP.
  • Adll-5ETel-GFP can be used to treat tumors or detect tumor cells in the blood circulation.
  • the expression of GFP in Adll-5ETel-GFP in human normal epithelial cells and cancer cells and the detection of CTCs indicate that dll-5ETel-GFP pair Tumor cells are very sensitive and can infect most tumor cells.
  • This vector can be used for the detection of tumor cells in circulating blood in a specific, sensitive and economical manner.
  • Figure 1 Schematic diagram of the construction of B-type recombinant human adenovirus vector Adll-5EP.
  • Figure 2 Comparison of the lytic potential of Adll-5EP on Adll-sensitive human tumor cells.
  • Figure 3 Comparison of the lytic potential of Adll-5EP against Adll-insensitive human tumor cells
  • the port indicates Ad5, ⁇ indicates Adll, and indicates Adll-5EP.
  • Figure 4 Growth curve of tumors treated with Ad5, Adll and Adll-5EP in a subcutaneously implanted allogeneic tumor model of MIA PaCa-2 cells.
  • Figure 5 Tumor clearance rate of animals treated with Ad5, Adll and Adll-5EP in a subcutaneously implanted allogeneic tumor model of MIA PaCa-2 cells;
  • FIG. 4 and Fig. 5 1 indicates PBS, 2 indicates Adll, 3 indicates Adll-5EP, and 4 indicates Ad5.
  • Figure 6 Schematic diagram of the construction of the shuttle vectors pSSENTel and pSSGFP and the replication-selective tumor-cracking adenovirus plasmid pAdll-5ETel-GFP.
  • FIG. 7 Comparison of expression of GFP in Adll-5ETel-GFP in human normal epithelial cells and cancer cells; in the figure, 1 is the image under white light, and 2 is the image under fluorescence.
  • Figure 8 Statistical histogram of the number of tumor cells in the blood detected by Adll-5 ETel-GFP.
  • Figure 9 Image of tumor cells in blood detected under Adll-5 ETel-GFP fluorescence microscope.
  • Example 1 Construction method of B-type recombinant human adenovirus vector Adll-5EP
  • the left arm sequence is 392 bp in front of the Adll genome
  • the right arm is the Ad5 E1A enhancer and the promoter sequence (195-559 bp) and the Adll E1A (568-I125 bp) are sequentially connected together, and the left arm and the right arm are respectively
  • the shuttle vector pSS-AlA7 was created by ligating to the multiple cloning site on both sides of pSS-Chl. pSS-AlA7 was digested with Pmel and purified.
  • the Pmel digested fragment was simultaneously homologously recombined with pAdll in BJ5183 cells, and then screened on agar plates containing ampicillin and chloramphenicol (concentration 100 mg/ml, 25 mg/ml, respectively).
  • the positive clones were digested with Swal to remove the anti-chloramphenicol gene expression cassette sequence, and finally pAdll-Ad5EP was generated.
  • pAdll-Ad5EP was linearized by Notl and transfected into 293 cells to produce adenovirus Adll-Ad5EP (see Figure 1).
  • Example 2 Adhesive potential of Ad5, Adll and Adll-5EP in Adll sensitive and insensitive human tumor cells.
  • Ad5 Adll in Adll-sensitive human tumor cells Capan-2, PaTu8988s, PC-3, MCF7, HT-29 and insensitive human tumor cells MIA PaCa-2, MDA-MB-23 HCT116, LNCaP and A549
  • Adll-5EP Ad5
  • the above 10 kinds of cells were made into cell suspension using 2% fetal bovine serum medium, inoculated in the center of 96-well plate, and after 14 to 18 hours, the virus was diluted.
  • the dO 4 pt/cell was used as the starting concentration, and the virus solution was diluted 10 times.
  • ⁇ /well was added to infect various cells in a 96-well plate, and MTS was added to detect their tumor-dissolving potential 6 days after infection.
  • Adll-5EP showed better cell killing ability than Ad5 in all Adll sensitive cell lines and produced stronger cytotoxicity than Adll (see Figure 2).
  • the performance of Adll-5EP was significantly improved (see Figure 3).
  • Adll-5EP showed high sensitivity in 90% (9 / 10) cell lines, indicating better efficacy against tumors than Ad5 and Adll.
  • Adll-5EP expanded the range of effective killing of tumor cells in vitro.
  • Example 3 Anti-tumor effects of Ad5, Adll and Adll-5EP in subcutaneously implanted allogeneic tumor models of MIA PaCa-2 cells.
  • Example 4 Construction method of B-type recombinant human adenovirus vector Adll-5ETel-GFP.
  • the nucleotide sequence of Swal-EcoRV-Fsel-Hindlll-Srfl-Sall-Bglll-Pmel) is ligated to the anti-chloramphenicol gene expression cassette sequence to generate pSS-CM, upstream (left arm) and downstream for homologous recombination ( The right arm) sequences are inserted into the multiple cloning sites on both sides, respectively, to construct a shuttle vector for recombination;
  • nucleotide sequence of Sbfl-Swal-EcoRV-Fsel-Hindlll-Srfl-Sall-Bglll-Pmel is linked to the anti-kanamycin gene expression cassette sequence to generate pSS-kna, upstream of the homologous recombination (left arm) And the downstream (right arm) sequence is inserted into the multiple cloning site on both sides of pSS-kna, respectively, to construct a shuttle vector for recombination;
  • the left arm sequence is 392 bp in front of the Adll genome
  • the Ad5 E1A enhancer (195-378 bp) in the right arm
  • the human TERT promoter (-714-Obp)
  • Adll E1A (568-1125 bp)
  • Fragment, two restriction enzyme sites Xbal and Ncol were introduced on both sides of the human TERT promoter, and the left and right arms were inserted into the corresponding sides of the pSS-Chl (Snabl and EcoRV), respectively, and finally pSSENTel was generated;
  • pSSGFP the left arm is a DNA fragment (Adll genomic DNA from 27301 to lj 27837 bp) and the EGFP gene is ligated through Ncol, a SnaBI site is introduced at 3' of EGFP, and the right arm is Adll genomic DNA from 28337 to 28920 bp.
  • the DNA fragment, the left arm and the right arm were blunt-ended into the corresponding Snabl and EcoRV sites on the corresponding sides of pSS-kna, and finally pSSGFP was generated;
  • pSSENTel and pSSGFP were digested and purified by Pmel, respectively. Two Pmel digested fragments were simultaneously with pAdll.
  • the homologous recombination was carried out in BJ5183 cells, and then screened on agar plates containing ampicillin, kanamycin and chloramphenicol (concentration: 100 mg/ml, 50 ug/ml, 25 mg/ml, respectively).
  • the positive clones were digested with Swal and Sbfl, respectively, to remove the anti-chloramphenicol gene expression cassette sequence and the kanamycin gene expression cassette sequence, and finally pAdll-5ETel-GFP was generated (see Fig. 6).
  • pAdll-5ETel-GFP was linearized by Notl and transfected into 293 cells to produce adenovirus Adll-5ETel-GFP.
  • Example 5 Expression of GFP in Adll-5ETel-GFP in human normal epithelial cells and cancer cells.
  • Adll-5ETel-GFP infected human pancreatic cancer cell line SUIT-2 and human normal bronchial epithelial cell line NHBE infected with concentration of lOOpfu/cell, and 24 hours later, the expression of GFP was observed by immunofluorescence microscopy. GFP is highly expressed in tumor cell SUIT-2 and lower in normal cell NHBE (see Figure 7), indicating that Adll-5ETel-GFP is sensitive to tumor cells.
  • Example 6 Adll-5 ETel-GFP detects circulating tumor cell CTCs.

Abstract

提供B型重组人腺病毒载体Ad11-5EP和Ad11-5ETel-GFP的构建方法及其应用。腺病毒载体Ad11-5EP的构建是利用同源重组将Ad5E1A编码序列上游的365bp段基因（包括Ad5E1A的增强子和启动子）置换到B型人腺病毒11亚型Ad11的相应区域，构建成载体Ad11-5EP。B型重组人腺病毒载体Ad11-5ETel-GFP的构建方法是由构建的穿梭载体pSSENTel、pSSGFP与Ad11-5EP基因组同时同源重组产生Ad11-5ETel-GFP。Ad11-5EP载体具有比野生型Ad11更强的溶肿瘤作用，增强了杀伤肿瘤细胞的能力。通过肿瘤生长和肿瘤清除率的测定表明，Ad11-5EP能有效降低肿瘤生长速度，且荷瘤鼠无瘤率显著好于Ad11；构建的Ad11-5ETel-GFP可用于治疗肿瘤或检测血液循环中的肿瘤细胞，可特异、敏感、经济的用于循环血液中的肿瘤细胞的检测。

Description

溶肿瘤能力增强的 B型人腺病毒 Adl l突变体的构建和应用 技术领域
[0002] 本发明涉及腺病毒载体及其应用， 特别是涉及 B 型重组人腺病毒载体 Adl l-5EP和 Adll-5ETel-GFP的构建方法及其应用。
技术背景
[0003] 近年来， 肿瘤病毒治疗特别是腺说病毒在肿瘤治疗方面的应用发展非常迅速。 2005年， E1B 55K和 E3B缺陷型腺病毒 (H101)首次在中国被批准用于治疗头颈部肿瘤， 然而， 腺病毒 来源的 H101 对某些恶性肿瘤的疗效很差， 比如发病高致死性强的胰腺癌。 限制肿瘤疗效的 书
原因之一是这些肿瘤低表达腺病毒受体 (CAR) , 而且， 静脉注射的腺病毒很快被抗体及补体 中和而灭活， 此外， 腺病毒还可以被 Kuffer细胞吞噬， 病毒 hexon蛋白与凝血因子 X结合， 然后转导肝细胞， 所以过量腺病毒容易导致明显的肝细胞毒性及肝损伤。
[0004] Adl l 是 B型人腺病毒的一种亚型。 在溶肿瘤治疗方面， Adll明显优越于 Ad5。 除了 CD46受体外， Adll还可以结合其他细胞表面受体 X。 Tuve等报道 Adll是 B腺病毒亚群中 唯一的既结合 CD46又结合 X的病毒， 说明 Adl l可以感染更广范的肿瘤细胞， 由此解决了 Ad5应用中病毒受体下调而导致感染率低的难题。 Adll较 Ad5另外一个优越之处在于人群中 Adl l的中和抗体较低为 10-31 %， Ad5是 45-90 %， 并且中间无交叉活性。 当静脉注射 Adll 到表达 CD46的转基因鼠时， 未发现明显的肝内转导和肝毒性现象。 而且， Adll能够高效转 导树突状细胞， 因此重组 Adll 可以表达肿瘤特异抗原， 通过增强免疫反应来更好的用于肿 瘤免疫治疗。
[0005] 关于 Ad5 以外的其它腺病毒血清型作为疫苗或基因转换载体的应用已经有了大量报 道。 但它们作为溶肿瘤病毒的研究才刚刚起步， 相关报道也不深入。 Sandberg等最近体内外 研究显示， Adl l能够有效地在前列腺癌细胞系 PC-3中转导、 复制并裂解， 但该作者没有与 通用的 Ad5进行比较。 Shashkova等应用人肿瘤细胞系体外试验及人前列腺癌细胞系 DU 145 体内研究， 比较了 Ad5、 Ad6、 Adl l和 Ad35之间的溶肿瘤效率， 发现 Ad5、 Ad6和 Adl l有 相似的抗肿瘤效果， 而 Ad35则无抗肿瘤作用。 重要的是， 仅仅 Ad5产生了肝脏毒性。 后来 人们因此建立了嵌合型溶肿瘤 Ad5(将 Ad5的纤毛替换成 B亚群腺病毒的)， 目的是使嵌合型 溶肿瘤 Ad5通过结合细胞膜上 CD46受体来提高抗肿瘤效果。但是与完整的 B亚群腺病毒相 比较， 这种方法仍不能克服针对 Ad5 hexon抗体的中和能力。
[0006] 循环肿瘤细胞（Circulating tumour cells,CTCs)数与肿瘤患者的临床分期、 疗效及短的 生存率相关。 肿瘤患者外周血液的 CTCs水平可作为监控和调整治疗及判断预后的依据。 这 就迫切需要一种特异的、敏感的方法去检测这些细胞。近年来，免疫细胞计数分析和定量 PCR 等方法的应用已经能够在血液中检测出少量的 CTCs，然而缺少特异的生物学标志物及高额的 检测费用限制了这些方法的普及应用。
[0007] 复制选择性溶肿瘤腺病毒是一类新的肿瘤治疗药物。 值得注意的是， 最近已经报导用 表达 GFP的复制选择性溶肿瘤腺病毒 Ad5在上亿外周血细胞中检测 CTCs。 然而， 肿瘤细胞 的遗传性变化是影响腺病毒感染的重要因素， 肿瘤细胞中低表达 CAR会显著降低 Ad5的感 染能力， 进而影响肿瘤细胞的阳性检出率。 并且发现， 除了已知的影响机制如 CAR低表达以 外， 另外一些肿瘤相关基因如 CEACAM6也能影响 Ad5进入到细胞核中， 进而减少了 Ad5 对肿瘤细胞的感染能力。 这些数据表明在一些肿瘤细胞中用 Ad5检测 CTCs的方法可能敏感 性较低。
发明内容
[0008] 本发明要解决的技术问题： 提供一种具有肿瘤靶向性和抗肿瘤效果的腺病毒载体
Adll-5EP, 还提供一种用于治疗肿瘤或检测血循环中肿瘤细胞的 B 型重组人腺病毒载体
Adll-5ETel-GFP。
[0009] 本发明的技术方案：
发明人首次在人肿瘤细胞系中体外比较了 Adll和 Ad5的抗肿瘤潜能，发现在 25个被检细胞 系中仅仅有 9个对 Adll敏感。其中 PC-3对 Ad5不敏感而对 Adll敏感。与 Ad5相比较， Adll 在体内明显抑制 PC-3细胞的皮下肿瘤生长， 并且提高了荷瘤动物的无瘤率。 同样， 用对 Ad5 敏感而对 Adll不敏感的 MIA PaCa-2细胞重复上述实验， Adll的抗肿瘤能力明显降低。
[0010] 尽管人肿瘤细胞中普遍高表达 Adll的受体，但野生型 Adll却不能有效地杀死这些肿 瘤细胞。 发明人对此进行了广泛而深入的研究。 用两种不同的方法， 发明人论证了与 Ad5相 比， 有更多的 Adll吸附在肿瘤细胞膜上， 这一结果与该细胞是否对 Adll敏感无关。 吸附的 Adll病毒颗粒能够有效地进入到细胞核内， 说明与 Ad5相比在病毒感染的早期阶段细胞核 内就有较高水平的 Adll。发明人调查比较了两种病毒在肿瘤细胞中的早期基因表达， 用特异 引物作定量 PCR方法来检测比较 E1A的 mRNA水平。 在所有这些细胞系中病毒感染 2小时 后， 发现较高水平的 Adll感染产生了较高水平的 E1A mRNA表达。 其中 Adll不敏感细胞 中 (MIA PaCa-2和 LNCaP)Ad5的 ElA mRNA表达水平在感染 2小时后明显降低。同样， Adll 敏感的 Capan-2和 PC-3细胞 Adll El A mRNA呈较高水平。 Adll El A mRNA直接影响着病 毒的复制， 所以 Adll E1A mRNA在 MIA PaCa-2和 LNCaP细胞中的下调会降低病毒复制水 平，相应地降低 hexon蛋白的合成能力，这一发现与最初观察到的 Adll低水平的病毒子的产 生及细胞毒性相一致。 这些结果表明 Adll 的复制及其杀伤能力与它的感染性无关， 而与它 的早期基因的增强子和启动子活性有关。
[0011] 为了解决上述问题， 本发明构建了一种肿瘤靶向性腺病毒载体 (Adll-5EP)。 实验表明 Adll-5EP是一个非常有用的基本载体， 可用于开发新的复制选择性溶肿瘤腺病毒， 以期治疗 更广泛的人类肿瘤。
[0012] 为了进一步探索该新型腺病毒载体的应用， 增强检测血循环肿瘤细胞的敏感性， 在 Adll-5EP基础上， Ad5启动子被人端粒酶基因启动子所取代， 然后通过同源重组产生了一个 表达信号基因的复制选择型腺病毒 (Adll-5ETel-GFP)。 由于端粒酶在 95 %人肿瘤细胞中高表 达，所以 Adll-5ETel-GFP只能选择性地在肿瘤细胞中复制并表达 GFP，而在正常上皮细胞中 没有活性。
[0013] 本发明的具体技术方案如下：
一种 B型重组人腺病毒载体 Adll-5EP的构建方法， 利用同源重组将 Ad5 E1A编码序列上游 的 365bp段基因，包括 Ad5 E1A的增强子和启动子，置换到 B型人腺病毒 11亚型 Adll的相 应区域， 构建成 B型重组人腺病毒载体 Adll-5EP。
[0014] 所述同源重组时， 左臂序列是 Adll基因组前 392bp段基因， 右臂为 Ad5 E1A增强子 及启动子序列的 195-559bp段基因和 Adll ElA 的 568-1125bp段基因， 按顺序连接到一起形 成片段， 左臂和右臂分别连接到 pSS-Chl 两侧多克隆位点上， 建立穿梭载体 pSS-AlA7 ; PSS-A1A7被 Pmel酶切并纯化， Pmel消化片段同时与 pAdll在 BJ5183细胞内进行同源重组， 然后在含氨苄青霉素及氯霉素两种抗生素的琼脂板上筛选； 阳性克隆分别经 Swal酶切，移除 抗氯霉素基因表达盒序列， 生成 pAdll-Ad5EP， pAdll-Ad5EP经 Notl酶切线性化后， 转染到 293细胞中产生腺病毒 Adll-5EP。
[0015] 所述氨苄青霉素、 氯霉素的浓度分别为 100mg/ml、 25mg/ml。
[0016] 利用 B型重组人腺病毒载体 Adll-5EP改造复制选择性溶肿瘤腺病毒的方法为下列方 法其中之一：
(1)删除腺病毒在正常细胞中生存必需的但在肿瘤细胞中不需要的基因 E1A CR2区域或抗凋 亡基因 E1B 21K;
(2)插入肿瘤特异启动子， 驱动 E1A基因的表达； (3)根据肿瘤细胞表面特异受体， 改造腺病毒的嗜细胞性；
(4)结合 MicroRNA技术， 赋予病毒选择性地在肿瘤细胞中进行复制。
[0017] 一种 B型重组人腺病毒载体 Adll-5ETel-GFP的构建方法， 包括以下步骤：
( 1 )先用两个不同的抗生素抗性表达盒构建载体 pSS-Chl和 pSS-kna, 在抗氯霉素基因表达 盒序列两侧引入 Swal酶切位点， 在抗卡那霉素基因表达盒序列两侧引入 sbfl酶切位位点；
(2) 从 pUC18克隆得到 pBR32复制的起始序列， 通过人工合成的包含多克隆位点的核苷酸 序列与抗氯霉素基因表达盒序列相连接产生 pSS-CM， 同源重组抗氯霉素基因表达盒序列上 游左臂序列和下游右臂序列， 将抗氯霉素上游左臂序列和下游右臂序列分别平端或粘端插入 到 pSS-Chl两侧的多克隆位点内， 构建重组用的穿梭载体；
(3) 从 pUC18克隆得到 pBR32复制的起始序列， 通过人工合成的包含多克隆位点的核苷酸 序列与抗卡那霉素基因表达盒序列相连接产生 pSS-kna,同源重组抗卡那霉素基因上游左臂序 列和下游右臂序列， 将抗卡那霉素基因上游左臂序列和下游右臂序列分别平端或粘端插入到 pSS-kna两侧的多克隆位点内， 构建重组用的穿梭载体；
(4) pSSENTel的构建， 左臂序列是 Adll基因组的前 392bp序列， 右臂为 Ad5 E1A增强子 的 195-378bp段基因、 人 TERT启动子的 -714-Obp段基因 禾卩 Adll E1A的 568-1125bp序列 按顺序连接在一起形成的片段， 同时在人 TERT启动子的两侧分别引入两个限制性酶切位点 Xbal和 NcoI， 左臂和右臂分别平端插入到 pSS-Chl的相应两侧 Snabl和 EcoRV, 最终生成 pSSENTel;
(5) pSSGFP的构建， 左臂为 Adll 基因组从 27301 至 lj 27837bp的 DNA片段与 EGFP基因 通过 Ncol的连接产物，并在 EGFP的 3'处引入 SnaBI位点；右臂是 Adll 基因组从 28337 到 28920bp的 DNA片段， 左臂和右臂分别平端插入到 pSS-kna两侧的 Snabl和 EcoRV位点， 最 终生成 pSSGFP;
(6) pSSENTel和 pSSGFP分别被 Pmel酶切并纯化， 两个 Pmel消化片段同时与 pAdll在 BJ5183细胞内进行同源重组， 然后在含氨苄青霉素、 卡那霉素及氯霉素三种抗生素的琼脂板 上进行筛选； 阳性克隆分别经 Swal和 Sbfl酶切， 移除抗氯霉素基因表达盒序列和卡那霉素 基因表达盒序列， 生成 pAdll-5ETel-GFP; pAdll-5ETel-GFP经 Notl酶切线性化后， 转染到 293细胞中产生腺病毒 Adll-5ETel-GFP。
[0018] 所述氨苄青霉素、 卡那霉素及氯霉素浓度依次为 100mg/ml、 50ug/ml、 25mg/ml。
[0019] 所述 pSSENTel中的 Tel序列能够被其他肿瘤特异基因的启动子所取代，生成一个肿瘤 特异的溶肿瘤腺病毒； 所述 pSSGFP中的 GFP序列能够被另外信号基因或治疗基因取代。 [0020] 所述 pSSGFP中的 Adll 18.5基因的启动子能够被更强的肿瘤特异启动子所取代。
[0021] 所述的 B型人腺病毒载体 Adll-5EP在用于治疗肿瘤中的应用。
[0022] 所述的 B型重组人腺病毒载体 Adll-5ETel-GFP在用于治疗肿瘤或检测血循环中肿瘤 细胞的应用。
[0023] 本发明的积极有益效果：
( 1 ) 本发明的肿瘤靶向性腺病毒载体 Adll-5EP， 是在野生型 Adll的基础上， 其 E1A的增 强子和启动子被 Ad5 E1A的增强子和启动子所取代得到的， 该载体比野生型 Adll有更强的 溶肿瘤作用， 从而增强了对肿瘤细胞的杀伤能力。
[0024] (2) 本发明的肿瘤靶向性腺病毒载体 Adll-5EP， 具有肿瘤靶向性和抗肿瘤效果。 通 过溶肿瘤潜能试验表明， Adll-5EP比 Ad5显示出更好的细胞杀伤能力， 比 Adll产生更强 的细胞毒性。 通过肿瘤生长和肿瘤清除率的测定表明， Adll-5EP能有效地降低肿瘤的生长 速度， 并且荷瘤鼠无瘤率显著好于 Adll。
[0025] (3)本发明的肿瘤靶向性腺病毒载体 Adll-5EP可作为一种肿瘤靶向性基因工程药物 治疗肿瘤， 将会产生较好的社会效益和经济效益。
(4) 本发明的 B 型重组人腺病毒载体 Adll-5ETel-GFP 的构建方法， 由构建的穿梭载体 pSSENTeK pSSGFP与 Adll-5EP基因组同时进行同源重组产生重组病毒 Adll-5ETel-GFP。 Adll-5ETel-GFP可用于治疗肿瘤或检测血循环中肿瘤细胞， 通过 Adll-5ETel-GFP中的 GFP 在人正常上皮细胞及癌细胞中的表达试验以及检测 CTCs的实验表明， dll-5ETel-GFP对肿瘤 细胞非常敏感， 它能够感染大多数肿瘤细胞， 用该载体可特异的、 敏感的、 经济的用于循环 血液中肿瘤细胞的检测。
附图说明
[0026] 图 1 : B型重组人腺病毒载体 Adll-5EP的构建示意图。
[0027] 图 2: Adll-5EP对 Adll敏感人肿瘤细胞的溶肿瘤潜能的比较。
[0028] 图 3: Adll-5EP对 Adll不敏感人肿瘤细胞的溶肿瘤潜能的比较；
图 2、 图 3中， 口表示 Ad5， 騸表示 Adll , 翻表示 Adll-5EP。
[0029] 图 4: MIA PaCa-2细胞皮下种植异体肿瘤模型中 Ad5、 Adll和 Adll-5EP处理后肿瘤 的生长曲线图。
[0030] 图 5: MIA PaCa-2细胞皮下种植异体肿瘤模型中 Ad5、 Adll和 Adll-5EP处理后动物 肿瘤清除率；
图 4、 图 5中， 1表示 PBS， 2表示 Adll , 3表示 Adll-5EP， 4表示 Ad5。 [0031] 图 6:穿梭载体 pSSENTel和 pSSGFP及复制选择性溶肿瘤腺病毒质粒 pAdll-5ETel-GFP 的构建过程示意图。
[0032] 图 7: Adll-5ETel-GFP中的 GFP在人正常上皮细胞及癌细胞中的表达比较图； 图中， 1为白光下图像， 2为荧光下图像。
[0033] 图 8: Adll-5ETel-GFP检测血液中肿瘤细胞数的统计柱状图。
[0034] 图 9: Adll-5ETel-GFP荧光显微镜下检测的血液中肿瘤细胞数图像。
具体实施方式
[0035] 以下通过实例对本发明进一步说明， 并不是限定本发明的保护范围， 本领域的技术人 员根据说明书可以对这些实施方案进行修改， 只要不脱离本发明的精神和范围都可以得到类 似或相同的结果， 均在本发明的保护范围之内。
[0036] 实例 1 : B型重组人腺病毒载体 Adll-5EP的构建方法
利用同源重组将 Ad5 E1A编码序列上游的 365bp段基因， 包括 Ad5 E1A的增强子和启动子， 置换到 B型人腺病毒 11亚型 Adll的相应区域， 构建成 B型重组人腺病毒载体 Adll-5EP。
[0037] 左臂序列是 Adll基因组前 392bp，右臂为 Ad5 E1A增强子及启动子序列 （195-559bp) 和 Adll E1A (568-I125bp)按顺序连接到一起的片段，左臂和右臂分别连接到 pSS-Chl两侧多 克隆位点上， 建立穿梭载体 pSS-AlA7。 pSS-AlA7被 Pmel酶切并纯化。 Pmel消化片段同时 与 pAdll在 BJ5183细胞内进行同源重组， 然后在含氨苄青霉素及氯霉素两种抗生素 （浓度 依次为 100mg/ml、 25mg/ml) 的琼脂板上筛选。 阳性克隆分别经 Swal酶切， 以移除抗氯霉素 基因表达盒序列， 最终生成 pAdll-Ad5EP。 pAdll-Ad5EP经 Notl酶切线性化后， 转染到 293 细胞中产生腺病毒 Adll-Ad5EP (参见图 1 )。
[0038] 实例 2: Ad5、 Adll和 Adll-5EP在 Adll敏感及不敏感人肿瘤细胞中的溶肿瘤潜能。
[0039] 在 Adll敏感人肿瘤细胞 Capan-2、 PaTu8988s、 PC-3、 MCF7、 HT-29和不敏感人肿瘤 细胞 MIA PaCa-2、 MDA-MB-23 HCT116、 LNCaP和 A549中对 Ad5、 Adll和 Adll-5EP 进行体外杀伤实验，用 2%胎牛血清的培养基将上述 10种细胞制成细胞悬液，接种于 96孔板 中央， 14〜18h之后， 倍比稀释病毒。 以 dO4 pt/细胞为起始浓度， 10倍比稀释病毒溶液， 加入 ΙΟμΙ/孔感染 96孔板中各种细胞， 感染后 6天加入 MTS检测它们的溶肿瘤潜能。
[0040] 结果显示：在所有 Adll敏感细胞系中， Adll-5EP比 Ad5显示出更好的细胞杀伤能力， 比 Adll产生更强的细胞毒性 （参见图 2)。 而在 Adll不敏感的细胞系中， Adll-5EP的性能 得到显著提高 （参见图 3)。 Adll-5EP在 90% (9 / 10) 的细胞系中都显示高敏感性， 说明比 Ad5和 Adll拥有更好的杀伤肿瘤的效力， Adll-5EP在体外扩大了有效杀伤肿瘤细胞的范围。 [0041] 实例 3: Ad5、 Adll和 Adll-5EP在 MIA PaCa-2细胞皮下种植异体肿瘤模型中的抗肿 瘤效果。
[0042] 在 BALA/c裸鼠 (n = 8 I组)右侧背部皮下接种 MIA PaCa-2细胞 （因为 MIA PaCa-2对 Adll不敏感而对 Ad5敏感）， 构建皮下移植瘤模型， 当肿瘤体积达到 180 mm3时， 第 1、 3、 5天瘤内分别注射 PBS或病毒 (Ad5、 Adll和 Adll-5EP， 1X101()病毒颗粒 /注射)， 观察肿瘤 生长和肿瘤清除率。 结果发现 Adll-5EP如 Ad5—样能有效地降低肿瘤的生长 （参见图 4)， 并且荷瘤鼠无瘤率显著好于 Adll处理组 （参见图 5)。
[0043] 实例 4: B型重组人腺病毒载体 Adll-5ETel-GFP的构建方法。
[0044] ( 1 )先用两个不同的抗生素抗性表达盒构建两个不同载体 pSS-Chl和 pSS-kna， 其中 抗氯霉素基因表达盒序列两侧引入 Swal酶切位点， 而抗卡那霉素基因表达盒序列两侧为 sbfl 切位位点；
(2) 从 pUC18克隆得到 pBR32复制起始序列， 然后通过人工合成的包含多克隆位点 ( Pmel-Kpnl-SacII-Notl-XhoI-Xbal-Sgfl-Snabl-Swal禾口
Swal-EcoRV-Fsel-Hindlll-Srfl-Sall-Bglll-Pmel)的核苷酸序列与抗氯霉素基因表达盒序列相连 接产生 pSS-CM， 同源重组需要的上游 (左臂)和下游 (右臂)序列分别平端或粘端插入到两侧的 多克隆位点内， 构建成重组用的穿梭载体；
(3) 从 pUC18克隆得到 pBR32复制起始序列， 然后通过人工合成的包含多克隆位点 ( Pmel-Kpnl-SacII-Notl-XhoI-Xbal-Sgfl-Snabl-Swal-Sbfl禾口
Sbfl-Swal-EcoRV-Fsel-Hindlll-Srfl-Sall-Bglll-Pmel)的核苷酸序列与抗卡那霉素基因表达盒序 列相连接产生 pSS-kna, 同源重组需要的上游 (左臂)和下游 (右臂)序列分别平端或粘端插入到 pSS-kna两侧的多克隆位点内构建成重组用的穿梭载体；
(4) pSSENTel, 左臂序列是 Adll基因组前 392bp， 右臂为 Ad5 E1A增强子 (195-378bp)、 人 TERT启动子 （-714-Obp) 和 Adll E1A (568-1125bp)按顺序连接到一起的片段， 在人 TERT 启动子两侧分别引入两个限制性酶切位点 Xbal和 Ncol，左臂和右臂分别平端插入到 pSS-Chl 的相应两侧 (Snabl和 EcoRV), 最终生成 pSSENTel;
(5) pSSGFP, 左臂为 DNA片段 (Adll 基因组 DNA从 27301 至 lj 27837bp)与 EGFP基因通 过 Ncol的连接产物，在 EGFP的 3'引入一个 SnaBI位点，右臂是 Adll 基因组 DNA从 28337 到 28920bp的 DNA片段， 左臂和右臂分别平端插入到 pSS-kna相应两侧的 Snabl和 EcoRV 位点， 最终生成 pSSGFP;
(6) pSSENTel和 pSSGFP分别被 Pmel酶切并纯化， 两个 Pmel消化片段同时与 pAdll在 BJ5183细胞内进行同源重组， 然后在含氨苄青霉素、 卡那霉素及氯霉素三种抗生素 （浓度依 次为 100mg/ml、 50ug/ml、 25mg/ml) 的琼脂板上筛选。 阳性克隆分别经 Swal和 Sbfl酶切， 以移除抗氯霉素基因表达盒序列和卡那霉素基因表达盒序列，最终生成 pAdll-5ETel-GFP (参 见图 6)。
[0045] pAdll-5ETel-GFP 经 Notl 酶切线性化后， 转染到 293 细胞中产生腺病毒 Adll-5ETel-GFP。
[0046] 实例 5: Adll-5ETel-GFP中的 GFP在人正常上皮细胞及癌细胞中的表达。
[0047] Adll-5ETel-GFP感染人胰腺癌细胞系 SUIT-2和人正常支气管上皮细胞系 NHBE (感 染浓度 lOOpfu/细胞）， 24小时后用免疫荧光显微镜观察 GFP的表达。 GFP在肿瘤细胞 SUIT-2 中呈现高表达， 而在正常细胞 NHBE中较低 （参见图 7)， 说明 Adll-5ETel-GFP对肿瘤细胞 敏感。
[0048] 实例 6: Adll-5ETel-GFP检测循环肿瘤细胞 CTCs。
[0049] 分别将 10、 25、 50、 100、 200个人胰腺癌细胞 SUIT-2混入 3ml血液中， 裂解红细胞 后离心收集有核细胞， 然后用 900 μ ΐ DMEM 培养基重悬有核细胞， 加入 lxlO4 pfu 的 Adll-5ETel-GFP, 孵育 24小时， 最后用免疫荧光显微镜计数 GFP阳性细胞 （参见图 8)。 外 周血细胞混入 0、 10、 100、 1000个人胰腺癌细胞 SUIT-2细胞 （感染浓度 lOOpfu/细胞）， 24 小时后用免疫荧光显微镜下观察荧光细胞 （参见图 9)， 结果表明 dll-5ETel-GFP对肿瘤细胞 非常敏感。
[0050]

Claims

权 利 要 求 书
1. 一种 B型重组人腺病毒载体 Adll-5EP的构建方法， 其特征是： 利用同源重组将 Ad5 ElA 编码序列上游的 365bp段基因， 包括 Ad5 E1A的增强子和启动子， 置换到 B型人腺病毒 11 亚型 Adll的相应区域， 构建成 B型重组人腺病毒载体 Adll-5EP。
2. 根据权利要求 1所述的构建方法， 其特征是： 所述同源重组时， 左臂序列是 Adll基因组 前 392bp段基因，右臂为 Ad5 E1A增强子及启动子序列的 195-559bp段基因和 Adll E1A 的 568-1125bp段基因， 按顺序连接到一起形成片段， 左臂和右臂分别连接到 pSS-Chl两侧多克 隆位点上， 建立穿梭载体 PSS-A1A7; pSS-AlA7被 Pmel酶切并纯化， Pmel消化片段同时与 pAdll在 BJ5183细胞内进行同源重组，然后在含氨苄青霉素及氯霉素两种抗生素的琼脂板上 筛选； 阳性克隆分别经 Swal 酶切， 移除抗氯霉素基因表达盒序列， 生成 pAdll-Ad5EP， PAdll-Ad5EP经 Notl酶切线性化后， 转染到 293细胞中产生腺病毒 Adll-5EP。
3. 根据权利要求 2 所述的构建方法， 其特征是： 所述氨苄青霉素、 氯霉素的浓度分别为 lOOmg/mK 25mg/ml。
4. 利用权利要求 1所述的 B型重组人腺病毒载体 Adll-5EP改造复制选择性溶肿瘤腺病毒的 方法为下列方法其中之一：
(1)删除腺病毒在正常细胞中生存必需但在肿瘤细胞中不需要的基因 E1A CR2区域或抗凋亡 基因 E1B 21K;
(2)插入肿瘤特异启动子， 驱动 E1A基因的表达；
(3)根据肿瘤细胞表面特异受体， 改造腺病毒的嗜细胞性；
(4)结合 MicroRNA技术， 赋予病毒选择性地在肿瘤细胞中进行复制。
5. —种 B型重组人腺病毒载体 Adll-5ETel-GFP的构建方法， 其特征是： 所述方法包括以下 步骤：
( 1 )先用两个不同的抗生素抗性表达盒构建载体 pSS-Chl和 pSS-kna, 在抗氯霉素基因表达 盒序列两侧引入 Swal酶切位点， 在抗卡那霉素基因表达盒序列两侧引入 sbfl酶切位位点；
(2) 从 pUC18克隆得到 pBR32复制的起始序列， 通过人工合成的包含多克隆位点的核苷酸 序列与抗氯霉素基因表达盒序列相连接产生 pSS-CM， 同源重组抗氯霉素基因表达盒序列上 游左臂序列和下游右臂序列， 将抗氯霉素上游左臂序列和下游右臂序列分别平端或粘端插入 到 pSS-Chl两侧的多克隆位点内， 构建重组用的穿梭载体；
(3) 从 pUC18克隆得到 pBR32复制的起始序列， 通过人工合成的包含多克隆位点的核苷酸 序列与抗卡那霉素基因表达盒序列相连接产生 pSS-kna,同源重组抗卡那霉素基因上游左臂序 列和下游右臂序列， 将抗卡那霉素基因上游左臂序列和下游右臂序列分别平端或粘端插入到 权 利 要 求 书
pSS-kna两侧的多克隆位点内， 构建重组用的穿梭载体；
(4) pSSENTel的构建， 左臂序列是 Adll基因组的前 392bp序列， 右臂为 Ad5 E1A增强子 的 195-378bp段基因、 人 TERT启动子的 -714-Obp段基因 禾卩 Adll E1A的 568-1125bp序列 按顺序连接在一起形成的片段， 同时在人 TERT启动子的两侧分别引入两个限制性酶切位点 Xbal和 NcoI， 左臂和右臂分别平端插入到 pSS-Chl的相应两侧 Snabl和 EcoRV, 最终生成 pSSENTel;
(5) pSSGFP的构建， 左臂为 Adll 基因组从 27301 至 lj 27837bp的 DNA片段与 EGFP基因 通过 Ncol的连接产物，并在 EGFP的 3'处引入 SnaBI位点；右臂是 Adll 基因组从 28337 到 28920bp的 DNA片段， 左臂和右臂分别平端插入到 pSS-kna两侧的 Snabl和 EcoRV位点， 最 终生成 pSSGFP;
(6) pSSENTel和 pSSGFP分别被 Pmel酶切并纯化， 两个 Pmel消化片段同时与 pAdll在 BJ5183细胞内进行同源重组， 然后在含氨苄青霉素、 卡那霉素及氯霉素三种抗生素的琼脂板 上进行筛选； 阳性克隆分别经 Swal和 Sbfl酶切， 移除抗氯霉素基因表达盒序列和卡那霉素 基因表达盒序列， 生成 pAdll-5ETel-GFP; pAdll-5ETel-GFP经 Notl酶切线性化后， 转染到 293细胞中产生腺病毒 Adll-5ETel-GFP。
6. 根据权利要求 5所述的构建方法， 其特征是： 所述氨苄青霉素、 卡那霉素及氯霉素浓度依 次为 100mg/ml、 50ug/ml、 25mg/ml。
7. 根据权利要求 5所述的构建方法， 其特征是： 所述 pSSENTel中的 Tel序列能够被其他肿 瘤特异基因的启动子所取代， 生成一个肿瘤特异的溶肿瘤腺病毒； 所述 pSSGFP中的 GFP序 列能够被另外信号基因或治疗基因取代。
8. 根据权利要求 5所述的构建方法， 其特征是： 所述 pSSGFP中的 Adll 18.5基因的启动子 能够被更强的肿瘤特异启动子所取代。
9. 权利要求 1的 B型人腺病毒载体 Adll-5EP在用于治疗肿瘤中的应用。
10. 权利要求 5的 B型重组人腺病毒载体 Adll-5ETel-GFP在用于治疗肿瘤或检测血循环中 肿瘤细胞的应用。
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