携带人卵泡刺激素受体的慢病毒载体疫苗的构建及其免疫效应检测

doi:10.13560/j.cnki.biotech.bull.1985.2016.03.024

摘要/Abstract

摘要： 制备携带卵泡刺激素受体的慢病毒载体疫苗, 并研究其对小鼠的免疫效应。将卵泡刺激素受体胞外区(fshr366)基因克隆到慢病毒载体上, 采用脂质体转染法将重组质粒转染至293T细胞中, 包装并产生含有目的基因的病毒颗粒；分别利用RT-PCR和Western blot检测感染病毒颗粒的293T细胞中fshr366 在mRNA水平及蛋白水平的表达情况；用携带fshr366的病毒颗粒单次腹腔免疫BALB/c小鼠, 分别在免疫0、14、21和28 d对小鼠眼眶采血, ELISA法检测免疫小鼠血清的特异性并测定抗体滴度。酶切和测序结果表明fshr366基因片段成功构建到慢病毒载体上。将包装产生的携带fshr366的慢病毒颗粒感染293T细胞后, RT-PCR和Western blot检测结果表明细胞在转录水平和蛋白水平均表达fshr基因。ELISA结果显示携带fshr366的慢病毒颗粒单次腹腔免疫小鼠后, 免疫14 d就激起了机体的体液免疫反应, 抗体滴度达到1:1 600。成功制备了携带卵泡刺激素受体的慢病毒载体疫苗, 其可以在小鼠体内激发FSHR抗原特异的早期持续性免疫反应。

关键词: 卵泡刺激素受体, 慢病毒载体, 疫苗, 体液免疫反应

Abstract: This work aims to prepare lentivirus vaccine with a follicle-stimulating hormone receptor(FSHR)and investigate the immunological effect of it on mice. The gene(fshr366)of extracellular region of FSHR was cloned into lentivirus vector. The recombinant plasmids were transfected into the 239T cells by the method of lipidosome transfection, and the virus particles(Lenti-FSHR366)with target gene were produced after encapsulation. The expressions of fshr366 mRNA and FSHR366 protein in 293T cells infected by Lenti-FSHR366 were detected by RT-PCR and Western blot. For evaluating the immunological effects, BALB/c mice were intraperitoneally inoculated with single immunization of fshr366-carring virus, collecting the blood samples from the orbits of mice at day 0, 14, 21 and 28 after immunization, then ELISA method was used to detect the specificity of the sera from immunized mice and determine the titer of the antibody. The RE digestion and DNA sequencing results showed the gene fragment of fshr366 was successfully cloned into lentivirus vector. After transfection to 293T cells with the encapsulated lentivirus particles carrying fshr366, the detection results by RT-PCR and Western blot indicated the expression of gene fshr in the cells at transcription and protein level. The results of ELISA revealed that the humoral immune response in mice rose on day 14 after single immunization through intraperitoneally injection of lentivirus particles carrying fshr366, the titer of antibody was 1:1600.

Key words: follicle-stimulating hormone receptor, lentivirus vector, vaccine, humoral response

马晓玲,刘红春,李江伟. 携带人卵泡刺激素受体的慢病毒载体疫苗的构建及其免疫效应检测[J]. 生物技术通报, 2016, 32(3): 148-154.

MA Xiao-ling, LIU Hong-chun, LI Jiang-wei. Development of a Lentivirus Vector-based Vaccine Carrying Follicle-stimulating Hormone Receptor and Assay of Its Immunological Effect[J]. Biotechnology Bulletin, 2016, 32(3): 148-154.

参考文献

[1]Pierce JG, Parsons TF. Glycoprotein hormones：structure and function[J]. Annual Review of Biochemistry, 1981, 50(1)：465-495.
[2]任春明, 字向东, 张重庆, 等. 卵泡刺激素的研究进展[J]. 科研前沿, 2006, 10(208)：10-13.
[3]Simoni M, Gromoll J, Nieschlag E. The Follicle-stimulating hormone receptor：Biochemistry, molecular biology, physiology, and pathop-hysiology 1[J]. Endocrine Reviews, 1997, 18(6)：739-773.
[4]Li Y, Ganta S, Cheng C, et al. FSH stimulates ovarian cancer cell growth by action on growth factor variant receptor[J]. Molecular and Cellular Endocrinology, 2007, 267(1)：26-37.
[5]Parrott JA, Doraiswamy V, Kim G, et al. Expression and actions of both the follicle stimulating hormone receptor and the luteinizing hormone receptor in normal ovarian surface epithelium and ovarian cancer[J]. Molecular and Cellular Endocrinology, 2001, 172(1)：213-222.
[6]Zheng W, Lu JJ, Luo F, et al. Ovarian epithelial tumor growth promotion by follicle-stimulating hormone and inhibition of the effect by luteinizing hormone[J]. Gynecologic Oncology, 2000, 76(1)：80-88.
[7]Radu A, Pichon C, Camparo P, et al. Expression of follicle-stimulating hormone receptor in tumor blood vessels[J]. New England Journal of Medicine, 2010, 363(17)：1621-1630.
[8]Stilley JA, Guan R, Duffy DM, et al. Signaling through FSH receptors on human umbilical vein endothelial cells promotes angiogenesis[J]. The Journal of Clinical Endocrinology & Metabolism, 2014, 99(5)：E813-E820.
[9]Gartrell BA, Tsao C, Galsky MD. The follicle-stimulating hormone receptor：a novel target in genitourinary malignancies[C]//Urologic Oncology：Seminars and Original Investigations. Elsevier, 2013, 31(8)：1403-1407.
[10] Ghinea N. A novel role for FSH receptor as a tumor endothelial cell marker[J]. Acta Endocrinologica(Buc), 2010, 6(4)：507-512.
[11]柏琦, 那鑫妮, 冯姗姗. 病毒载体的发展历程及现状[J]. 中国医药指南, 2011, 9(23)：224-225.
[12]Palmowski MJ, Lopes L, Ikeda Y, et al. Intravenous injection of a lentiviral vector encoding NY-ESO-1 induces an effective CTL response[J]. The Journal of Immunology, 2004, 172(3)：1582-1587.
[13]李明峰, 候静, 熊伟民. 携带AFP基因慢病毒载体制备并感染树突状细胞的实验研究[J]. 细胞与分子免疫学杂志, 2009, 25(9)：791-793.
[14]罗望, 张泓, 许淼, 等. 慢病毒—基因转移的潜在新载体[J]. 江苏药学与临床研究, 2006, 14(6)：366-371.
[15]Esslinger C, Chapatte L, Finke D, et al. In vivo administration of a lentiviral vaccine targets DCs and induces efficient CD8(+)T cell responses[J]. J Clin Invest, 2003, 111：1673-1681.
[16]Breckpot K, Dullaers M, Bonehill A, et al. Lentivirally transduced dendritic cells as a tool for cancer immunotherapy[J]. J Gene Med, 2003, 5：654-667.
[17]Esslinger C, Romero P, MacDonald HR. Efficient transduction of dendritic cells and induction of a T-cell response by thirdgeneration lentivectors[J]. Hum Gene Ther, 2002, 13：1091-1100.
[18]Palmowski MJ, Lopes L, Ikeda Y, et al. Intravenous injection of a lentiviral vector encoding NY-ESO-1 induces an effective CTL response[J]. J Immunol, 2004, 172：1582-1587.
[19]Iglesias MC, Frenkiel MP, Mollier K, et al. A single immunization with a minute dose of a lentiviral vector-based vaccine is highly effective at eliciting protective humoral immunity against West Nile virus[J]. The Journal of Gene Medicine, 2006, 8(3)：265-274.
[20]Wunseh A, AhdaY, Banaz-Yasar F, et al. Single-nucleotide poly-morphisms in the promoter region influence the expression of the human follicle-stimulating Hormone receptor[J]. Fertil Steril, 2005, 84(2)：446-453．
[21]Zhang X, Chen J, Zheng Y, et al. Follicle-stimulating hormone peptide can facilitate paclitaxel nanoparticles to target ovarian carcinoma in vivo[J]. Cancer Research, 2009, 69(16)：6506-6514.
[22]董俊. 基于卵泡刺激素素受体介导的靶向治疗卵巢癌实验研究[D]. 上海：复旦大学, 2011.
[23]Stilley JA, Guan R, Duffy DM, et al. Signaling through FSH recept-ors on human umbilical vein endothelial cells promotes angiogene-sis[J]. The Journal of Clinical Endocrinology & Metabolism, 2014, 99(5)：E813-E820.