Schlafen2基因在细胞中的表达及其生物学意义
|
摘要
研究背景:Schlafen(Slfn)家族基因是Schwarz DA等于1998年发现的参与调控胸腺发育的基因,目前对其所知甚少,所有国内外公开发表的文献只有6篇,仅有的研究表明,该家族基因缺陷可引起一种胚胎致死性表型-DDK综合症。Schlafen家族基因作为一类新发现的基因,其在肿瘤发生发展中的作用尚不清楚。Schlafen1(Slfn1)基因可通过拮抗对Cyclin D1的诱导,从而导致细胞周期终止于G1期。对于Schlafen2(Slfn2)的基因功能目前还不清楚,而且尚未建成能够稳定表达Slfn2基因的细胞系,使其进一步研究收到了限制。因此,探索Slfn2的基因功能对于细胞在基因水平上的研究是一项崭新的课题,这势必为人类基因组的研究进展提供新的视角,从而展开更加多元化的系统研究。
目的:(1)将Slfn2基因转入NIH/Swiss小鼠胚胎来源的纤维母细胞样细胞NIH/3T3中,通过绿色荧光蛋白标记观察细胞中Slfn2基因的表达及分布;(2)建立Slfn2基因稳定表达的细胞系以进一步研究其生物学功能;(3)克隆Slfn2基因启动子区序列,为今后构建双报告基因表达载体、检测荧光素酶的相对活性从而确定和研究小鼠Slfn2启动子的大致区域和相对活性奠定基础。
方法:(1)克隆Slfn2基因全编码区序列;(2)构建pEGFP-Slfn2及PCI-neo.Slfn2真核表达载体;(3)瞬时转染pEGFP-Slfn2载体及其空载体对照至NIH/3T3细胞,激光共聚焦显微技术观察细胞中Slfn2基因的表达及分布;(4)用脂质体Lipofectamine~(TM)2000分别转染pEGFP-Slfn2与pCI-Sifn2.neo及相应空载体对照至NIH/3T3细胞,G418筛选获得抵制的细胞株;(5)用Northern blot核酸分子杂交技术进一步鉴定Slfn2在各细胞株中的表达情况:(6)利用细胞生长曲线和Transwell侵袭实验检测Slfn2对细胞增殖及迁移能力的影响。(7)克隆Slfn2基因启动子区序列,以便后期将该序列重组至荧光素酶报告载体pGL3-Basic上,连接形成真核表达载体,与pRL-CMV共转染至NIH/3T3细胞,通过双荧光素酶活性的测定来确定和研究小鼠Slfn2启动子的大致区域和相对活性。
结果:
1.RT-PCR扩增出Slfn2全编码区序列片段,测序分析结果表明与Genebank中登录的Slfn2序列一致。
2.所构建的pEGFP-Slfn2及PCI-Slfn2.neo真核表达载体经酶切鉴定并测序,结果表明Slfn2基因与表达载体连接方向正确且无突变,可以进行转染。
3.瞬时转染对照及pEGFP-Slfn2质粒,激光共聚焦显微镜下NIH/3T3细胞中核、浆均可见到绿色荧光,说明Slfn2在细胞核、浆均有表达。
4.稳定转染pEGFP-Slfn2载体及pCI-Slfn2.neo及其相应的空载体对照于NIH/3T3细胞中,G418筛选得到抵制的细胞克隆;用Northern blot核酸分子杂交技术鉴定并筛选出表达Slfn2的阳性细胞株;扩增培养建立细胞系。实验中获得的阳性细胞株中Slfn2基因均可正确、稳定地表达。
5.绘制转染后细胞生长曲线:稳定表达Slfn2基因的细胞增殖速度明显减慢。
6.利用Transwell肿瘤细胞侵袭实验分析和检测转染后NIH/3T3细胞的迁移能力:Sifn2基因稳定表达的肿瘤细胞迁移侵袭能力显著降低。
7.PCR扩增出Slfn2启动子区序列片段,测序分析结果表明与Genebank中登录的Slfn2序列一致。
结论:(1)Slfn2的真核表达载体构建成功,首次获得了Slfn2基因稳定表达的细胞株。(2)Slfn2基因的稳定表达可明显影响肿瘤细胞的生物学特性,对肿瘤的生长增殖和迁移能力起负性调控作用,有效减缓肿瘤增殖速度,限制肿瘤蔓延扩散,即Slfn2基因的表达在肿瘤的发生发展及其转移过程中发挥着重要的作用,可能是一个潜在的肿瘤抑制基因。(3)成功克隆出Slfn2基因启动子区序列,可用于Slfn2启动子大致区域和相对活性的进一步研究。
Background:Schlafen family genes which participates regulation of thoracic gland development were discovered by Schwarz DA in 1998.There are only six literatures published,so it is very few about them to know so far.In these results,embryo-fatal phaenotype-DDK syndrome would be caused when the family gene is defective.As a group of genes which were discovered newly,the role of Schlafen family genes in tumor formation and development is indefinite.Schlafenl(Slfn1)could lead cell cycle to end in G1 stage by resisting the derivation of Cyclin D1.It is not clear to know the Gene function of Slfn2 at present.There's no cell line which is established to express Slfn2 gene stably,which restricts the development of further researching.For this reason,it is a brand-new subject to explore the effects of Slfn2 gene.It will be bound to provide a fresh view angle for advancement of researching human genome.So we could carry out more polynary and systematical researches.
Objective:(1)Slfn2 was transiently transfected into NIH/3T3 cells.To observe the position of Slfn2 gene directly with laser scanning confocal microscopy.Because the expressing proteins of Slfn2 were marked by green fluorescent protein(GFP).NIH/3T3 cell is a kind of mouse embryo-source blastoid cells.(2)To establish cell line which could express Schlafen2(Slfn2)gene stably and to elucidate biological functions of the gene.(3)To done the promoter region sequences of Slfn2.It will lay the foundation of prospective work.In future studies,the promoter domain and relative activity of Slfn2 gene in mice will be determined by some means,including to construct double report gene expressing vector and to detect the relative activity of luciferase.
Methods:(1)To clone the whole coding region sequences of Slfn2 gene.(2)To construct pEGFPSlfn2 and pCI-Slfn2.neo eukaryotic expressing vectors.(3)pEGFP-Slfn2 vector and contrast control were transiently transfected into NIH/3T3 cells to observe the position of Slfn2 gene directly with laser scanning confocal microscopy.(4)pEGFP- Slfn2 and pCI-Slfn2.neo were stably transfected into NIH/3T3 cells respectively with Lipofectamine~(TM)2000,and G418 screening was used to obtain resistant cell strains.(5)The expression of Slfn2 was measured by Northern blot.(6) Growth curve and Transwell were utilized to analyze the effects of Slfn2 on cell proliferation and migration.(7)To clone the promoter region sequences of Slfn2 in order to connect the sequences to pGL3-Basic vector.The eukaryotic expressing vector which formed in the above-mentioned step will be cotransfect with pRL-CMV into NIH/3T3 cells.Therefore,we could determine the promoter domain and relative activity of Slfn2 gene in mice by detecting the relative activity of luciferase in future.
Results:
1.The whole coding region sequences of Slfn2 were gained by RT-PCR.The result of DNA sequencing proved that the products were consonant with the sequences of Slfn2 in Genebank.
2.Restriction enzymes were used on the pEGFP-Slfn2 and pCI-Slfn2.neo eukaryotic expressing vectors.By means of this,we identified the vectors.Subsequently,the results of DNA sequencing told us that the Slfn2 genes were correct and competent for transfection.The genes were used in carrier conjugation.
3.pEGFP-Slfn2 vector and contrast control were transiently transfected into NIH/3T3 cells to observe the position of Slfn2 gene directly with laser scanning confocal microscopy.There're green fluorescence in cell nucleus and cytolymph.The photos demonstrated that the proteins expressed by the gene were detected in cell nucleus and cytolymph.
4.3T3 cells were stably transfected with the plasmid expressing Slfn2 and corresponding empty vectors.By screened with G418,we obtained resistant cell clones.The expression of Slfn2 was measured by Northern blot.Chose the positive cells and cultivated them to establish cell line.The results showed that the expressions of Slfn2 in these cell strains were correct and stable.
5.Drawing growth curve of the cells which were transfected.The results showed that the cell proliferation was inhibited markedly when the expression of Slfn2 was stable in these cells.
6.Transwell experiment was used to analyze and detect the migration of negative controls and the cell clones stably expressing Slfn2.The results told us that the capability of migration was degrade strikingly when the expression of Slfn2 was stable in these cells.
7.The promoter region sequences of Slfn2 were gained by PCR.The result of DNA sequencing showed that the sequences were identical with the one of Slfn2 in Genebank.
Conclusions:(1)The eukaryotic expressing vectors containing Slfn2 were constructed successfully.Moreover,cell line which could express Slfn2 gene stably was established by first time. (2)Biological character of tumor cells would change obviously when Slfn2 expressed stably.It plays a part in nagtive regulation of cell proliferation and migration.Proliferation and migration were inhibited markedly when the expression of Slfn2 was stable in these cells.It is possible that Slfn2 is a potential tumor suppressor gene.Therefore,the expression of the gene is essential at tumor formation and development.(3)The promoter region sequences of Slfn2 were amplified successfully, which can be used to determine the promoter domain and relative activity of Slfn2 gene in mice in future.
目的:(1)将Slfn2基因转入NIH/Swiss小鼠胚胎来源的纤维母细胞样细胞NIH/3T3中,通过绿色荧光蛋白标记观察细胞中Slfn2基因的表达及分布;(2)建立Slfn2基因稳定表达的细胞系以进一步研究其生物学功能;(3)克隆Slfn2基因启动子区序列,为今后构建双报告基因表达载体、检测荧光素酶的相对活性从而确定和研究小鼠Slfn2启动子的大致区域和相对活性奠定基础。
方法:(1)克隆Slfn2基因全编码区序列;(2)构建pEGFP-Slfn2及PCI-neo.Slfn2真核表达载体;(3)瞬时转染pEGFP-Slfn2载体及其空载体对照至NIH/3T3细胞,激光共聚焦显微技术观察细胞中Slfn2基因的表达及分布;(4)用脂质体Lipofectamine~(TM)2000分别转染pEGFP-Slfn2与pCI-Sifn2.neo及相应空载体对照至NIH/3T3细胞,G418筛选获得抵制的细胞株;(5)用Northern blot核酸分子杂交技术进一步鉴定Slfn2在各细胞株中的表达情况:(6)利用细胞生长曲线和Transwell侵袭实验检测Slfn2对细胞增殖及迁移能力的影响。(7)克隆Slfn2基因启动子区序列,以便后期将该序列重组至荧光素酶报告载体pGL3-Basic上,连接形成真核表达载体,与pRL-CMV共转染至NIH/3T3细胞,通过双荧光素酶活性的测定来确定和研究小鼠Slfn2启动子的大致区域和相对活性。
结果:
1.RT-PCR扩增出Slfn2全编码区序列片段,测序分析结果表明与Genebank中登录的Slfn2序列一致。
2.所构建的pEGFP-Slfn2及PCI-Slfn2.neo真核表达载体经酶切鉴定并测序,结果表明Slfn2基因与表达载体连接方向正确且无突变,可以进行转染。
3.瞬时转染对照及pEGFP-Slfn2质粒,激光共聚焦显微镜下NIH/3T3细胞中核、浆均可见到绿色荧光,说明Slfn2在细胞核、浆均有表达。
4.稳定转染pEGFP-Slfn2载体及pCI-Slfn2.neo及其相应的空载体对照于NIH/3T3细胞中,G418筛选得到抵制的细胞克隆;用Northern blot核酸分子杂交技术鉴定并筛选出表达Slfn2的阳性细胞株;扩增培养建立细胞系。实验中获得的阳性细胞株中Slfn2基因均可正确、稳定地表达。
5.绘制转染后细胞生长曲线:稳定表达Slfn2基因的细胞增殖速度明显减慢。
6.利用Transwell肿瘤细胞侵袭实验分析和检测转染后NIH/3T3细胞的迁移能力:Sifn2基因稳定表达的肿瘤细胞迁移侵袭能力显著降低。
7.PCR扩增出Slfn2启动子区序列片段,测序分析结果表明与Genebank中登录的Slfn2序列一致。
结论:(1)Slfn2的真核表达载体构建成功,首次获得了Slfn2基因稳定表达的细胞株。(2)Slfn2基因的稳定表达可明显影响肿瘤细胞的生物学特性,对肿瘤的生长增殖和迁移能力起负性调控作用,有效减缓肿瘤增殖速度,限制肿瘤蔓延扩散,即Slfn2基因的表达在肿瘤的发生发展及其转移过程中发挥着重要的作用,可能是一个潜在的肿瘤抑制基因。(3)成功克隆出Slfn2基因启动子区序列,可用于Slfn2启动子大致区域和相对活性的进一步研究。
Background:Schlafen family genes which participates regulation of thoracic gland development were discovered by Schwarz DA in 1998.There are only six literatures published,so it is very few about them to know so far.In these results,embryo-fatal phaenotype-DDK syndrome would be caused when the family gene is defective.As a group of genes which were discovered newly,the role of Schlafen family genes in tumor formation and development is indefinite.Schlafenl(Slfn1)could lead cell cycle to end in G1 stage by resisting the derivation of Cyclin D1.It is not clear to know the Gene function of Slfn2 at present.There's no cell line which is established to express Slfn2 gene stably,which restricts the development of further researching.For this reason,it is a brand-new subject to explore the effects of Slfn2 gene.It will be bound to provide a fresh view angle for advancement of researching human genome.So we could carry out more polynary and systematical researches.
Objective:(1)Slfn2 was transiently transfected into NIH/3T3 cells.To observe the position of Slfn2 gene directly with laser scanning confocal microscopy.Because the expressing proteins of Slfn2 were marked by green fluorescent protein(GFP).NIH/3T3 cell is a kind of mouse embryo-source blastoid cells.(2)To establish cell line which could express Schlafen2(Slfn2)gene stably and to elucidate biological functions of the gene.(3)To done the promoter region sequences of Slfn2.It will lay the foundation of prospective work.In future studies,the promoter domain and relative activity of Slfn2 gene in mice will be determined by some means,including to construct double report gene expressing vector and to detect the relative activity of luciferase.
Methods:(1)To clone the whole coding region sequences of Slfn2 gene.(2)To construct pEGFPSlfn2 and pCI-Slfn2.neo eukaryotic expressing vectors.(3)pEGFP-Slfn2 vector and contrast control were transiently transfected into NIH/3T3 cells to observe the position of Slfn2 gene directly with laser scanning confocal microscopy.(4)pEGFP- Slfn2 and pCI-Slfn2.neo were stably transfected into NIH/3T3 cells respectively with Lipofectamine~(TM)2000,and G418 screening was used to obtain resistant cell strains.(5)The expression of Slfn2 was measured by Northern blot.(6) Growth curve and Transwell were utilized to analyze the effects of Slfn2 on cell proliferation and migration.(7)To clone the promoter region sequences of Slfn2 in order to connect the sequences to pGL3-Basic vector.The eukaryotic expressing vector which formed in the above-mentioned step will be cotransfect with pRL-CMV into NIH/3T3 cells.Therefore,we could determine the promoter domain and relative activity of Slfn2 gene in mice by detecting the relative activity of luciferase in future.
Results:
1.The whole coding region sequences of Slfn2 were gained by RT-PCR.The result of DNA sequencing proved that the products were consonant with the sequences of Slfn2 in Genebank.
2.Restriction enzymes were used on the pEGFP-Slfn2 and pCI-Slfn2.neo eukaryotic expressing vectors.By means of this,we identified the vectors.Subsequently,the results of DNA sequencing told us that the Slfn2 genes were correct and competent for transfection.The genes were used in carrier conjugation.
3.pEGFP-Slfn2 vector and contrast control were transiently transfected into NIH/3T3 cells to observe the position of Slfn2 gene directly with laser scanning confocal microscopy.There're green fluorescence in cell nucleus and cytolymph.The photos demonstrated that the proteins expressed by the gene were detected in cell nucleus and cytolymph.
4.3T3 cells were stably transfected with the plasmid expressing Slfn2 and corresponding empty vectors.By screened with G418,we obtained resistant cell clones.The expression of Slfn2 was measured by Northern blot.Chose the positive cells and cultivated them to establish cell line.The results showed that the expressions of Slfn2 in these cell strains were correct and stable.
5.Drawing growth curve of the cells which were transfected.The results showed that the cell proliferation was inhibited markedly when the expression of Slfn2 was stable in these cells.
6.Transwell experiment was used to analyze and detect the migration of negative controls and the cell clones stably expressing Slfn2.The results told us that the capability of migration was degrade strikingly when the expression of Slfn2 was stable in these cells.
7.The promoter region sequences of Slfn2 were gained by PCR.The result of DNA sequencing showed that the sequences were identical with the one of Slfn2 in Genebank.
Conclusions:(1)The eukaryotic expressing vectors containing Slfn2 were constructed successfully.Moreover,cell line which could express Slfn2 gene stably was established by first time. (2)Biological character of tumor cells would change obviously when Slfn2 expressed stably.It plays a part in nagtive regulation of cell proliferation and migration.Proliferation and migration were inhibited markedly when the expression of Slfn2 was stable in these cells.It is possible that Slfn2 is a potential tumor suppressor gene.Therefore,the expression of the gene is essential at tumor formation and development.(3)The promoter region sequences of Slfn2 were amplified successfully, which can be used to determine the promoter domain and relative activity of Slfn2 gene in mice in future.
引文
[1] Schwarz DA, Katayama CD, Hedrick SM. Schlafen, a Flew family of growth regulatory genes that affect thymocyte development [J]. Immunity, 1998, 9(5): 657-668.
[2] Bell TA, de la Casa-Esperon E, Doherty HE, et al. The paternal gene of the DDK syndrome maps to the Schlafen gene cluster on mouse chromosome 11. Genetics, 2006, 172(1): 41 1-423.
[3] Brady G, Boggan L, Bowie A, et al. Schlafen-1 causes a cell cycle arrest by inhibiting induction of cyclin D1 [J]. J Biol Chem, 2005,280(35): 30723-30734
[4] Ghaffar H, Sahin F, Sanchez-CM, et al. LKB1 protein expression in the evo- lution of glandular neoplasia of the lung [J]. Clin Cancer Res, 2003,9(8): 2998-3003.
[5] Sohn WJ, Kim D, Leeand KW, et al. Novel transcriptional regulation of the schlafen-2 gene in macrophages in response to TLR-triggered stimulation. [J]. Molecular Immunology, 2007, 44(13): 3273-3282.
[6] Eskra L, Mathison A, Splitter G. Microarray analysis of mRNA levels from RAW 264.7 macrophages infected with Brucella abortus[J]. Infect Immun, 2003, 71:1125-1133.
[7] Schurr JR, Young E, Byrne P, et al .Central role of Toll-like receptor 4 signaling and host defense in experimental pneumonia caused by gram-negative bacteria[J]. Infect Immun, 2005,73: 532-545.
[8] Smith J A, Schmechel SC, Raghavan A, et al .Reovirus induces and benefits from an integrated cellular stress response[J]. Virol, 2006 ,80:2019-2033.
[9] Fujikado N, Saijo S, Iwakura Y. Identification of arthritis-related gene clusters by microarray analysis of two independent mouse models for rheumatoid arthritis [J]. Arthritis Res Ther,2006,8(4):R100.
[10] Li G, Hu Y, Huo Y, Liu M, et al. PTEN deletion leads to up-regulation of a secreted growth factor pleiotrophin[J]. J Biol Chem, 2006, 281(16): 10663-10668.
[11] Serrano M, Lin AW, Currach M, et al. Oncogenic ras provokes premature cell senesc- ence associated with accumulation of p53 and pl6 INK4a [J]. Cell, 1997, 88:593-596.
[12]Franke TF,Yang SL,Chart TO,et al.The protein kinase encoded by Akt protoinco- gene is a target of the PDGF-activated phosphatidylinositol 3 kinase[J].Cell,1995,81:727-731.
[13]Gualano RC,Pryor MJ,Cauchi MR,et al.Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length eDNA[J].J Gen Virol,1998,79(3):437-446.
[14]Liliental J,Moon S Y,Lesche R,et al.Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Racl and Cdc42 GTPases[J].Curr Biol,2000,10(7):401-404.
[15]霍艳英,胡迎春,周乔丹,et al.多效生长因子Pleiotrophin基因沉默后的基因表达谱初步分析.中国生物化学与分子生物学报[J].2007,23(5):351-356.
[16]Kinney RM,Butrapet S,Chang GJ,et al.Construction of infectious cDNA clones for dengue vires:strain 16681 and its attenuated vaccine derivative,strain PDK-53[J].Virology,1997,230(3):300-308.
[17]Polo S,Ketner G,Levis R,et al.Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast[J].J Virol,1997,71(7):5366-5374.
[18]Chalife M,Tu Y,Euskirchen G,et a.l Green fluorescen protein as amarker for gene expression.Science,1994,263(5 148):802.
[19]张峰,任燕,陆长德.绿色荧光蛋白及其应用.生命科学,1999,11(2):61
[2] Bell TA, de la Casa-Esperon E, Doherty HE, et al. The paternal gene of the DDK syndrome maps to the Schlafen gene cluster on mouse chromosome 11. Genetics, 2006, 172(1): 41 1-423.
[3] Brady G, Boggan L, Bowie A, et al. Schlafen-1 causes a cell cycle arrest by inhibiting induction of cyclin D1 [J]. J Biol Chem, 2005,280(35): 30723-30734
[4] Ghaffar H, Sahin F, Sanchez-CM, et al. LKB1 protein expression in the evo- lution of glandular neoplasia of the lung [J]. Clin Cancer Res, 2003,9(8): 2998-3003.
[5] Sohn WJ, Kim D, Leeand KW, et al. Novel transcriptional regulation of the schlafen-2 gene in macrophages in response to TLR-triggered stimulation. [J]. Molecular Immunology, 2007, 44(13): 3273-3282.
[6] Eskra L, Mathison A, Splitter G. Microarray analysis of mRNA levels from RAW 264.7 macrophages infected with Brucella abortus[J]. Infect Immun, 2003, 71:1125-1133.
[7] Schurr JR, Young E, Byrne P, et al .Central role of Toll-like receptor 4 signaling and host defense in experimental pneumonia caused by gram-negative bacteria[J]. Infect Immun, 2005,73: 532-545.
[8] Smith J A, Schmechel SC, Raghavan A, et al .Reovirus induces and benefits from an integrated cellular stress response[J]. Virol, 2006 ,80:2019-2033.
[9] Fujikado N, Saijo S, Iwakura Y. Identification of arthritis-related gene clusters by microarray analysis of two independent mouse models for rheumatoid arthritis [J]. Arthritis Res Ther,2006,8(4):R100.
[10] Li G, Hu Y, Huo Y, Liu M, et al. PTEN deletion leads to up-regulation of a secreted growth factor pleiotrophin[J]. J Biol Chem, 2006, 281(16): 10663-10668.
[11] Serrano M, Lin AW, Currach M, et al. Oncogenic ras provokes premature cell senesc- ence associated with accumulation of p53 and pl6 INK4a [J]. Cell, 1997, 88:593-596.
[12]Franke TF,Yang SL,Chart TO,et al.The protein kinase encoded by Akt protoinco- gene is a target of the PDGF-activated phosphatidylinositol 3 kinase[J].Cell,1995,81:727-731.
[13]Gualano RC,Pryor MJ,Cauchi MR,et al.Identification of a major determinant of mouse neurovirulence of dengue virus type 2 using stably cloned genomic-length eDNA[J].J Gen Virol,1998,79(3):437-446.
[14]Liliental J,Moon S Y,Lesche R,et al.Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Racl and Cdc42 GTPases[J].Curr Biol,2000,10(7):401-404.
[15]霍艳英,胡迎春,周乔丹,et al.多效生长因子Pleiotrophin基因沉默后的基因表达谱初步分析.中国生物化学与分子生物学报[J].2007,23(5):351-356.
[16]Kinney RM,Butrapet S,Chang GJ,et al.Construction of infectious cDNA clones for dengue vires:strain 16681 and its attenuated vaccine derivative,strain PDK-53[J].Virology,1997,230(3):300-308.
[17]Polo S,Ketner G,Levis R,et al.Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast[J].J Virol,1997,71(7):5366-5374.
[18]Chalife M,Tu Y,Euskirchen G,et a.l Green fluorescen protein as amarker for gene expression.Science,1994,263(5 148):802.
[19]张峰,任燕,陆长德.绿色荧光蛋白及其应用.生命科学,1999,11(2):61