牛PDGFRA基因克隆及过表达载体构建的研究
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摘要
为了研究血小板衍生生长因子受体α(platelet-derived growth factor receptor alpha,PDGFRA)基因对牛睾丸支持细胞(sertoli cells,SCs)的增殖作用,试验采用RT-PCR方法扩增PDGFRA基因CDs区,TA克隆并测序,然后用T4 DNA连接酶构建pBI-CMV3-PDGFRA过表达载体,并进行酶切和测序鉴定,最后应用转染试剂FuGene HD将过表达载体转染到SCs中进行荧光观察。结果表明:PCR扩增得到的PDGFRA基因CDs全长为3 276 bp,经NCBI BLAST生物信息学软件对比TA克隆测序序列同源性为100%,构建的pBI-CMV3-PDGFRA过表达载体经鉴定得到的片段和序列结果正确,转染SCs 24 h后在荧光显微镜下可观察到绿色荧光蛋白。说明试验成功构建出过表达载体pBI-CMV3-PDGFRA,并在SCs中正常表达。
In order to study the proliferation effect of platelet-derived growth factor receptor alpha(PDGFRA) gene in sertoli cells(SCs), PDGFRA gene CDs area was amplificated by RT-PCR firstly. And TA cloning method and sequence analysis were used.Then pBI-CMV3-PDGFRA over expression vector was constructed using T4 DNA Ligase. Next, verification was implemented through enzyme digestion and sequencing. At last, FuGene HD reagent was used to transfect the over-expression vector into SCs. The results showed that the PDGFRA gene CDs with a total length of 3 276 bp was obtained by PCR amplification. The homology of TA-clone sequence with NCBI BLAST bioinformatics software was 100%. Identification results of pBI-CMV3-PDGFRA over-expression vector were correct by enzyme digestion and sequencing. After 24 h of transfection, the green fluorescent protein in SCs were observed under fluorescence microscopy. It indicated that the pBI-CMV3-PDGFRA over-expression vector was successfully constructed and normally expressed in SCs.
In order to study the proliferation effect of platelet-derived growth factor receptor alpha(PDGFRA) gene in sertoli cells(SCs), PDGFRA gene CDs area was amplificated by RT-PCR firstly. And TA cloning method and sequence analysis were used.Then pBI-CMV3-PDGFRA over expression vector was constructed using T4 DNA Ligase. Next, verification was implemented through enzyme digestion and sequencing. At last, FuGene HD reagent was used to transfect the over-expression vector into SCs. The results showed that the PDGFRA gene CDs with a total length of 3 276 bp was obtained by PCR amplification. The homology of TA-clone sequence with NCBI BLAST bioinformatics software was 100%. Identification results of pBI-CMV3-PDGFRA over-expression vector were correct by enzyme digestion and sequencing. After 24 h of transfection, the green fluorescent protein in SCs were observed under fluorescence microscopy. It indicated that the pBI-CMV3-PDGFRA over-expression vector was successfully constructed and normally expressed in SCs.
引文
[1] BASCIANI S,MARIANI S,ARIZZI M,et al. Expression of platelet-derived growth factor (PDGF) in the epididymis and analysis of the epididymal development in PDGF-A, PDGF-B, and PDGF receptor beta deficient mice[J].Biol Reprod,2004,70(1):168-177.
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[3] 郭农建,徐从高.血小板衍生生长因子及与某些肿瘤的关系[J].国外医学(肿瘤学分册),1993,20(6):326-329.
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[5] 孙蕾. 靶向PDGF-A RNA干涉抑制增生性玻璃体视网膜病变中RPE细胞增殖的实验研究[D].长春:吉林大学,2008.
[6] BAUMAN J E, EATON K D,MARTINS R G. Antagonism of platelet-derived growth factor receptor in nonsmallcell lung cancer: rationale and investigations[J]. Clin Cancer Res,2007,13(15 Pt 2):s4632-s4636.
[7] MATEI D, EMERSON R E, LAI Y C, et al. Autocrine activation of PDGFRalpha promotes the progression of ovarian cancer[J]. Oncogene,2006,25(14):2060-2069.
[8] SEKIDO R,LOVELL-BADGE R. Genetic control of testis development[J].Sex Dev,2012,7(1/2/3):21-32.
[9] VALEIR C, SCHTEINGART H F, REY R A.The prepubertal testis: biomarkers and functions[J].Curr Opin Endocrinol Diabetes Obes,2013,20(3):224-233.
[10] ORTH J M, GUNSALUS G L, LAMPERTI A A. Evidence from sertoli cell-depleted rats indicates that spermatid number in adults depends on numbers of sertoli cells produced during perinatal development[J]. Endocrinology, 1988,122(3):787-794.
[11] SHARP R M, MCKINNELL C, KIVLIN C, et al. Proliferation and functional maturation of sertoli cells, and their relevance to disorders of testis function in adulthood[J].Reproduction,2003, 1259(6):769-784.
[12] 高一,秦立红,房希碧,等.PDGFRA基因对牛未成熟支持细胞体外增殖的影响[J].吉林农业大学学报,2018,40(3):352-357.
[13] 杨润军,张路培,许尚忠,等.牛 FADD 基因的克隆分析及其真核表达载体的构建[J].西北农林科技大学学报,2009,37(2):15-26.
[14] 汪旭莹.支持细胞在生精细胞凋亡过程中的重要作用[J].细胞生物学杂志,2002,24(6):325-328.
[15] GRIMALDI P, DI GIACOMO D, GEREMIA R.The endocannab-inoid system and spermatogenesis[J]. Front Endocrinol (Laus-anne),2013, 4:192.
[16] 江小华.支持细胞依赖的结构和功能对小鼠生殖细胞发育影响的研究[D].合肥:中国科学技术大学,2014.
[17] MENEGAZZO M, ZUCCARELLO D, LUCA G, et al.Improvements in human sperm quality by long-term in vitro co-culture with isolated porcine sertoli cells[J]. Hum Reprod,2011,26(10):2598-2605.
[18] MA C, SONG H, GUAN K, et al. Characterization of swine testicular cell line as immature porcine sertoli cell line[J].In Vitro Cell Dev Biol Anim,2016,52(4):427-433.
[19] CHAN F, LIU Y, SUN H, et al.Distribution and possible role of PDGF-AA and PDGFR-α in the gastrointestinal tract of adult guinea pigs[J]. Virchows Arch,2010, 457(3):381-388.
[20] 赵伟.猪不同发育阶段睾丸组织中miR-34b/c及预测靶基因差异表达研究[D].长春:吉林大学,2013.
[2] FREDRIKSSON L, LI H, ERIKSSON U. The PDGF family: four gene products form five dimeric isoforms [J].Cytokine Growth Factor Rev,2004,15(4): 197-204.
[3] 郭农建,徐从高.血小板衍生生长因子及与某些肿瘤的关系[J].国外医学(肿瘤学分册),1993,20(6):326-329.
[4] 刘兰,白玛多吉.血小板衍生生长因子PDGF与肿瘤血管生成的研究进展[J].西藏大学学报(自然科学版),2011,26(2):92-97.
[5] 孙蕾. 靶向PDGF-A RNA干涉抑制增生性玻璃体视网膜病变中RPE细胞增殖的实验研究[D].长春:吉林大学,2008.
[6] BAUMAN J E, EATON K D,MARTINS R G. Antagonism of platelet-derived growth factor receptor in nonsmallcell lung cancer: rationale and investigations[J]. Clin Cancer Res,2007,13(15 Pt 2):s4632-s4636.
[7] MATEI D, EMERSON R E, LAI Y C, et al. Autocrine activation of PDGFRalpha promotes the progression of ovarian cancer[J]. Oncogene,2006,25(14):2060-2069.
[8] SEKIDO R,LOVELL-BADGE R. Genetic control of testis development[J].Sex Dev,2012,7(1/2/3):21-32.
[9] VALEIR C, SCHTEINGART H F, REY R A.The prepubertal testis: biomarkers and functions[J].Curr Opin Endocrinol Diabetes Obes,2013,20(3):224-233.
[10] ORTH J M, GUNSALUS G L, LAMPERTI A A. Evidence from sertoli cell-depleted rats indicates that spermatid number in adults depends on numbers of sertoli cells produced during perinatal development[J]. Endocrinology, 1988,122(3):787-794.
[11] SHARP R M, MCKINNELL C, KIVLIN C, et al. Proliferation and functional maturation of sertoli cells, and their relevance to disorders of testis function in adulthood[J].Reproduction,2003, 1259(6):769-784.
[12] 高一,秦立红,房希碧,等.PDGFRA基因对牛未成熟支持细胞体外增殖的影响[J].吉林农业大学学报,2018,40(3):352-357.
[13] 杨润军,张路培,许尚忠,等.牛 FADD 基因的克隆分析及其真核表达载体的构建[J].西北农林科技大学学报,2009,37(2):15-26.
[14] 汪旭莹.支持细胞在生精细胞凋亡过程中的重要作用[J].细胞生物学杂志,2002,24(6):325-328.
[15] GRIMALDI P, DI GIACOMO D, GEREMIA R.The endocannab-inoid system and spermatogenesis[J]. Front Endocrinol (Laus-anne),2013, 4:192.
[16] 江小华.支持细胞依赖的结构和功能对小鼠生殖细胞发育影响的研究[D].合肥:中国科学技术大学,2014.
[17] MENEGAZZO M, ZUCCARELLO D, LUCA G, et al.Improvements in human sperm quality by long-term in vitro co-culture with isolated porcine sertoli cells[J]. Hum Reprod,2011,26(10):2598-2605.
[18] MA C, SONG H, GUAN K, et al. Characterization of swine testicular cell line as immature porcine sertoli cell line[J].In Vitro Cell Dev Biol Anim,2016,52(4):427-433.
[19] CHAN F, LIU Y, SUN H, et al.Distribution and possible role of PDGF-AA and PDGFR-α in the gastrointestinal tract of adult guinea pigs[J]. Virchows Arch,2010, 457(3):381-388.
[20] 赵伟.猪不同发育阶段睾丸组织中miR-34b/c及预测靶基因差异表达研究[D].长春:吉林大学,2013.