HCBP6蛋白与HCVcore蛋白相互作用的研究
摘要
目的：
在前期工作的基础上,进一步应用免疫共沉淀方法验证HCVcore蛋白与HCBP6蛋白之间的相互作用。并应用激光共聚焦技术了解HCVcore蛋白与HCBP6蛋白的亚细胞定位。为进一步明确HCBP6蛋白功能奠定基础。
方法：
1、用实时荧光PCR方法检测HCBP6在7种癌细胞系的表达,选择较高表达的细胞系用于随后的蛋白之间相互作用的真核水平的研究。
2、将真核表达质粒pcDNA3.1-hcvcore、pcDNA3.1/myc-his(-)-hcbp6、pcDNA3.1及pcDNA3.1/myc-his(-)瞬时转染HepG2细胞,应用免疫共沉淀方法验证HCVcore蛋白与HCBP6蛋白之间的相互作用。
3、构建HCBP6的绿色荧光表达载体和HCVcore的红色荧光表达载体,转染HepG2细胞,模拟该蛋白在细胞中的表达,在激光共聚焦显微镜下观察HCBP6及HCVcore蛋白的亚细胞定位。
结果：
1、HCBP6在Capan2细胞中表达最高,依次为HepG2、Hela、Huh7、7721、7701与7402。
2、pcDNA3.1-hcvcore及pcDNA3.1/myc-his(-)-hcbp6在HepG2细胞中单独转染和共转染都获得成功。
3、HCBP6的绿色荧光表达载体和HCVcore的红色荧光表达载体在HepG2细胞中均得到表达。激光共聚焦显微镜观察到HCBP6蛋白和HCVcore蛋白均主要定位于细胞浆中。
结论：
1、HCVcore为丙型肝炎病毒核心蛋白,研究它与HCBP6之间的作用应该选择一种肝癌细胞系。HCBP6在HepG2细胞系中表达水平较高,又因为HepG2是肝癌细胞系所以选择HepG2为后续实验的细胞系。
2、HCBP6蛋白和HCVcore蛋白之间确实存在相互作用。
3、HCBP6蛋白和HCVcore蛋白在细胞内均分布于细胞浆中,分布的部位相同,再次证明这两种蛋白之间存在相互作用。并初步表明HCBP6蛋白在细胞浆中发挥生物学活性。
Objective:
To testify the interaction between HCBP6 and HCVcore by CO-IP. And to understand the subcellular localization of HCBP6 and HCVcore by LSCM(laser scan confocal microscopy). To identify the function of HCBP6 is useful to establish search foundation.
Methods:
1.Real-time PCR was adopted to compare different quantitative expression of HCBP6 from 7 cell lines.The cell line with higher expression was selected for subsequent experiment to evalute interaction between proteins on eukaryotic cell level.
2.HepG2 cells was transfected with pcDNA3.1-hcvcore、pcDNA 3.1/myc-his(-)-hcbp6、pcDNA3.1and pcDNA3.1/myc-his(-), sepa-rately. The interaction between HCBP6 and HCVcore was evaluated by CO-IP.
3.The GFP expressing vector of HCBP6 and pDsRed expressing vector of HCVcore were constructed after confirming by DNA sequence analysis and restriction enzyme digestion. The plasmids were transfected into HepG2 cell lines by lipofectamine, respectively. Subcellular localization was detected by the LSCM (laser scan confocal microscopy).
Results:
1.The expression of HCBP6 in different cell lines from highest to low-est is Capan2、HepG2、Hela、Huh7、7721、7701 and 7402.
2.Both co-transfection and independent transfection of pcDNA 3.1-hcvcore and pcDNA3.1/myc-his(-)-hcbp6 to HepG2 are proved to be successful.
3.The GFP expressing vector of HCBP6 and pDsRed expressing vector of HCVcore were successful expressing in the HepG2 cell lines. They are both detected in cytoplasm.
Conclusion:
1.HCVcore is the hepatitis C virus core protein.To study the role with HCBP6 should choose one between the hepatocellelar carcinoma cell lines.The expression of HCBP6 in HepG2 cell line is high and HepG2 is one kind of hepatoma carcinoma cell lines. HepG2 cell line was chosen for the following experiment.
2.Interaction between HCBP6 and HCVcore are proved.
3.Both HCBP6 and HCVcore are detected in cytoplasm. Subcellular localization establish the premise for interaction between HCBP6 and HCVcore.HCBP6 educe biologic activity in cytoplasm.
在前期工作的基础上,进一步应用免疫共沉淀方法验证HCVcore蛋白与HCBP6蛋白之间的相互作用。并应用激光共聚焦技术了解HCVcore蛋白与HCBP6蛋白的亚细胞定位。为进一步明确HCBP6蛋白功能奠定基础。
方法：
1、用实时荧光PCR方法检测HCBP6在7种癌细胞系的表达,选择较高表达的细胞系用于随后的蛋白之间相互作用的真核水平的研究。
2、将真核表达质粒pcDNA3.1-hcvcore、pcDNA3.1/myc-his(-)-hcbp6、pcDNA3.1及pcDNA3.1/myc-his(-)瞬时转染HepG2细胞,应用免疫共沉淀方法验证HCVcore蛋白与HCBP6蛋白之间的相互作用。
3、构建HCBP6的绿色荧光表达载体和HCVcore的红色荧光表达载体,转染HepG2细胞,模拟该蛋白在细胞中的表达,在激光共聚焦显微镜下观察HCBP6及HCVcore蛋白的亚细胞定位。
结果：
1、HCBP6在Capan2细胞中表达最高,依次为HepG2、Hela、Huh7、7721、7701与7402。
2、pcDNA3.1-hcvcore及pcDNA3.1/myc-his(-)-hcbp6在HepG2细胞中单独转染和共转染都获得成功。
3、HCBP6的绿色荧光表达载体和HCVcore的红色荧光表达载体在HepG2细胞中均得到表达。激光共聚焦显微镜观察到HCBP6蛋白和HCVcore蛋白均主要定位于细胞浆中。
结论：
1、HCVcore为丙型肝炎病毒核心蛋白,研究它与HCBP6之间的作用应该选择一种肝癌细胞系。HCBP6在HepG2细胞系中表达水平较高,又因为HepG2是肝癌细胞系所以选择HepG2为后续实验的细胞系。
2、HCBP6蛋白和HCVcore蛋白之间确实存在相互作用。
3、HCBP6蛋白和HCVcore蛋白在细胞内均分布于细胞浆中,分布的部位相同,再次证明这两种蛋白之间存在相互作用。并初步表明HCBP6蛋白在细胞浆中发挥生物学活性。
Objective:
To testify the interaction between HCBP6 and HCVcore by CO-IP. And to understand the subcellular localization of HCBP6 and HCVcore by LSCM(laser scan confocal microscopy). To identify the function of HCBP6 is useful to establish search foundation.
Methods:
1.Real-time PCR was adopted to compare different quantitative expression of HCBP6 from 7 cell lines.The cell line with higher expression was selected for subsequent experiment to evalute interaction between proteins on eukaryotic cell level.
2.HepG2 cells was transfected with pcDNA3.1-hcvcore、pcDNA 3.1/myc-his(-)-hcbp6、pcDNA3.1and pcDNA3.1/myc-his(-), sepa-rately. The interaction between HCBP6 and HCVcore was evaluated by CO-IP.
3.The GFP expressing vector of HCBP6 and pDsRed expressing vector of HCVcore were constructed after confirming by DNA sequence analysis and restriction enzyme digestion. The plasmids were transfected into HepG2 cell lines by lipofectamine, respectively. Subcellular localization was detected by the LSCM (laser scan confocal microscopy).
Results:
1.The expression of HCBP6 in different cell lines from highest to low-est is Capan2、HepG2、Hela、Huh7、7721、7701 and 7402.
2.Both co-transfection and independent transfection of pcDNA 3.1-hcvcore and pcDNA3.1/myc-his(-)-hcbp6 to HepG2 are proved to be successful.
3.The GFP expressing vector of HCBP6 and pDsRed expressing vector of HCVcore were successful expressing in the HepG2 cell lines. They are both detected in cytoplasm.
Conclusion:
1.HCVcore is the hepatitis C virus core protein.To study the role with HCBP6 should choose one between the hepatocellelar carcinoma cell lines.The expression of HCBP6 in HepG2 cell line is high and HepG2 is one kind of hepatoma carcinoma cell lines. HepG2 cell line was chosen for the following experiment.
2.Interaction between HCBP6 and HCVcore are proved.
3.Both HCBP6 and HCVcore are detected in cytoplasm. Subcellular localization establish the premise for interaction between HCBP6 and HCVcore.HCBP6 educe biologic activity in cytoplasm.
引文
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2. Johannessen M, Moller S, Hansen T, et al. The muhifunctionalroles of the four and a haf-LIM only protein FHL [J]. Cell Mol Life Sci.2006.63:268-284
3. Lin J,Arlinghaus R.Activated c-Abl tyrosine in malignant solid tumors [J]. Oncogene,2008,27:4385-4391
4. 李楠等.激光扫描共聚焦显微术.人民军医出版社,1997,37-59
5. 免疫荧光基础—实验新原理及临床新应用.第二版.北京：人民卫生出版社
6. Durrenberger M B,Handschin S,Conde Petit B,et al. Visualization of Food Structure by Confocal Laser Scanning Microscopy (CLSM) [J] Lebensm Wissu Technol,2001,34(1):11.
7. 张旭,徐维奇.激光扫描共聚焦显微镜技术的发展及应用[J].现代科学仪器,2001,2：21.
8. 郗昕,姜泗长,方耀云.激光扫描共聚焦显微镜的原理与生物学应用[J]中国体视学与图像分析,1996,1(3,4)：74.
9. 激光共聚焦扫描显微镜简介河北医科大学学报2002年9月第23卷第5期
10.韩贻仁.分子细胞生物学.第二版.上海科学出版社,P1
11.赵启韬,苗俊英.激光共聚焦显微镜在生物医学研究中的应用[J]北京生物医学工程,2003,22(1)：52.
12. Lamprecht A, Schfer U F, Lehr CM. Characterization of microcapsules by confocal laser scanning microscopy:structure, capsule wall composition and encapsulation rate [J].Eur J Pharm Biopharm,2000,49:1.
13. Win K Y, Feng S. Effect s of particle size and surface coatingon cellular uptake of polymeric nanoparticles for oral delivery of anticancer drug [J] Biomaterials,2005,26:2713.
14. Torchilin V P. Fluorescence microscopy to follow the targeting of liposomes and micelles to cells and their intracellular fate [J]. Adv Drug Deliv Rev 2005,57:95.
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