Cloning of rat serum amyloid P component gene and preparation of its recombinant adenovirus vector

LOU Xiao-li; et al

doi:10.13406/j.cnki.cyxb.2010.04.006

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Cloning of rat serum amyloid P component gene and preparation of its recombinant adenovirus vector

更新时间：2024-01-17

- Cloning of rat serum amyloid P component gene and preparation of its recombinant adenovirus vector
- Journal of Chongqing Medical University Vol. 35, Issue 4, Pages: 579-581(2010)
- 作者机构：
  
  重庆医科大学儿童医院呼吸中心
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.2010.04.006
  CLC： R341
- Published：2010，
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[1]娄小丽,黄英,舒畅,陈洁,罗庆,王莉佳,吴雪郡.大鼠血清淀粉样物质P(SAP)基因的克隆及重组腺病毒制备[J].重庆医科大学学报,2010,35(04):579-581.

LOU Xiao-li, et al. Cloning of rat serum amyloid P component gene and preparation of its recombinant adenovirus vector. [J]. Journal of Chongqing Medical University 35(4):579-581(2010)
[1]娄小丽,黄英,舒畅,陈洁,罗庆,王莉佳,吴雪郡.大鼠血清淀粉样物质P(SAP)基因的克隆及重组腺病毒制备[J].重庆医科大学学报,2010,35(04):579-581. DOI： 10.13406/j.cnki.cyxb.2010.04.006.

LOU Xiao-li, et al. Cloning of rat serum amyloid P component gene and preparation of its recombinant adenovirus vector. [J]. Journal of Chongqing Medical University 35(4):579-581(2010) DOI： 10.13406/j.cnki.cyxb.2010.04.006.

摘要

目的:制备能够表达大鼠血清淀粉样物质P（Serum amyloid P component

SAP）的重组腺病毒。方法:采用RT-PCR扩增的方法获取大鼠SAP

并将该基因采用定向克隆的方法克隆进入穿梭质粒pAdTrace-TO4中

进一步采用同源重组的方法将该质粒和腺病毒骨架质粒Adeasy-1在大肠杆菌BJ5183中同源重组

然后用脂质体介导的方法导入包装细胞HEK293中

产生表达目的基因的重组腺病毒

扩增病毒后用倍比稀释法测定其滴度。结果:通过PCR技术从重组穿梭质粒和重组腺病毒质粒中扩增出700bp左右插入片段

测序结果和基因库中的基因序列一致。扩增后的重组腺病毒滴度为2.8×108pfu/ml。结论:成功构建了携带SAP基因的重组腺病毒载体Adeasy-1-SAP

为探讨SAP对肺纤维化的作用及其机制打下基础。

Abstract

Objective:To construct the recombinant adenovirus vector carrying rat serum amyloid P component(SAP)as preparation for later use.Methods:Rat SAP was obtained by using RT-PCR amplification and then cloned into the shutter plasmid pAdTrace-TO4.Subsequently

this newly constructed plasmid pAdTrace-TO4-SAP was followed by homologous recombination with bone plasmid pAdEasy-1 in BJ5183.Recombinant adenovirus was obtained after being packaged in human embryonic kidney cells HEK293 by lipofectAmineTM2000 mediation.After amplification the recombined adenovirus

we determined its titer by end-point dilution assay.Results:In recombinant shuttle plasmid and recombinant adenovirus

a fragment of 700 bp detected by PCR was identical with that included in Genbank.The titer of recombinant adenovirus reached 2.8×108 pfu/ml.Conclusion:The recombinant adenovirus containing SAP gene was constructed successfully.This will provides a good basis for further study about the influence of SAP on lung fibrosis.

关键词

血清淀粉样物质P腺病毒肺纤维化分子克隆

Keywords

SAPadenovirusLungfibrosisClone

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