Construction of eukaryotic expression vector and package of lentivirus vector encoding prostate cancer antigen 3

刘晓军; 王娜; 姚旭东; 曹达龙

doi:10.3969/j.issn.1007-3969.2013.11.001

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Construction of eukaryotic expression vector and package of lentivirus vector encoding prostate cancer antigen 3

更新时间：2025-12-31

- Construction of eukaryotic expression vector and package of lentivirus vector encoding prostate cancer antigen 3
- China Oncology Vol. 23, Issue 11, Pages: 857-862(2013)
- 作者机构：
  
  1. 复旦大学附属肿瘤医院泌尿外科，复旦大学上海医学院肿瘤学系,上海,200032
  2. 复旦大学附属肿瘤医院肿瘤研究所，复旦大学上海医学院肿瘤学系,上海,200032
- 作者简介：
- 基金信息：
- DOI：10.3969/j.issn.1007-3969.2013.11.001
  CLC：
- Published Online：18 February 2014，
  
  Published：18 February 2013
- 稿件说明：
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刘晓军,王娜,姚旭东,曹达龙. 前列腺特异抗原3慢病毒表达载体的构建与包装[J]. 中国癌症杂志, 2013, 23(11): 857-862.

刘晓军, 王娜, 姚旭东, et al. Construction of eukaryotic expression vector and package of lentivirus vector encoding prostate cancer antigen 3[J]. China Oncology, 2013, 23(11): 857-862.
刘晓军,王娜,姚旭东,曹达龙. 前列腺特异抗原3慢病毒表达载体的构建与包装[J]. 中国癌症杂志, 2013, 23(11): 857-862. DOI： 10.3969/j.issn.1007-3969.2013.11.001.

刘晓军, 王娜, 姚旭东, et al. Construction of eukaryotic expression vector and package of lentivirus vector encoding prostate cancer antigen 3[J]. China Oncology, 2013, 23(11): 857-862. DOI： 10.3969/j.issn.1007-3969.2013.11.001.

摘要

背景与目的：前列腺癌抗原3(prostate cancer antigen 3，PCA3)作为一种长链非编码RNA(lncRNA)已被证实在前列腺癌中高度特异性表达，暗示其可能在前列腺癌中发挥重要作用，为深入研究，我们将完整的PCA3基因转入真核表达载体，并构建慢病毒包装系统。方法：从前列腺癌细胞株LNCaP细胞中提取总RNA，运用重叠延伸方法扩增到PCA3基因，测序正确后定向接入真核表达载体pCDH-CMV-MCS-EF1-copGFP，酶切鉴定并测序正确后进行慢病毒包装和滴度测定。结果：经PCR鉴定及测序，PCA3表达质粒序列与Gene Bank序列进行Blast比对分析，同源性为99.8%，PCA3慢病毒滴度测定为2×108。结论：PCA3成功插入真核表达载体并完成了慢病毒包装，为深入研究PCA3基因在前列腺细胞中的作用奠定了基础，进而为探索前列腺癌的治疗提供了新的途径。

Abstract

Background and purpose: The increased of specific expression of prostate cancer antigen 3 (PCA3)

as one of long non-coding RNA

has been observed in prostate cancer

indicating that PCA3 may contribute to the development of prostate cancer. To further study its roles in prostate cancer

we construct a lentivirus expression vector carrying the whole PCA3. Methods: PCA3 was amplified from prostate cancer cell line LNCaP by reverse transcriptase polymerase chain reaction (RT-PCR). After the sequence was proved to be correct

we recombined the pCDH-CMV-MCS-EF1-copGFP-PCA3. After transformation into E.coli cells

the candidate clones were identified by PCR amplifying

restricting enzyme digestion analysis and DNA sequencing

and the viral titer was determined. Results: Through the Blast analysis software

we compared the results of PCA3 sequence amplified by PCR with GeneBank sequence

finding that the homology is 99.8%. The lentivirus vector was constructed successfully

and the virus in the supernatant reached a titer of 2*108. Conclusion: The successful construction of the lentivirus vector encoding PCA3 not only lays the foundation for the further research into the effect of PCA3 gene on the prostate cancer but also provides a new therapy for advanced prostate cancer.

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