|
|
Prokaryotic Expression,Purification and Preparation of Monoclonal Antibodies of Cold Inducible RNA-binding Protein in BALB/C Mice |
1.College of Animal Science and Technology, Heilongjiang August First Land Reclamation University, Daqing 163319, China
2.Harbin Veterinary Research Insititue, Chinese Academy of Agricultural Sciences, Harbin 15000,China |
|
|
Abstract The CIRP gene of mouse was amplified from testis total RNA of BALB/C mouse by RT-PCR. PCR product was cloned into the pGEM-T vector to construct recombinant plasmid pGEM-CIRP for sequencing. The correct CIRP sequence was inserted into pQE-30-Xa to construct expression vector pQE-30-CIRP.It was transformed into host strain E.coli M15 and was induced by IPTG. It shows efficient expression in the E.coli by SDS-PAGE detected. The expression product exists in inclusion body. CIRP fusion protein was purified by Ni-NTA affinity chromatography and electroelution. The purified CIRP protein was used to immune BALB/C mice three times. Result of indirect ELISA shows that antibody titers of serum was 1∶105. The hybridoma technique was adopted to fusion. Positive hybridoma cell was screened by indirect ELISA and 3 times subcloning. Western blot was used to indentify McAb specificity. Indirect ELISA was used to identify McAb IgG subclasses and determine titer of McAb cell supernatants and ascites. 1A6, 2D3, 3C5 and 4C4 4 hybridoma cell line of stable secretion CIRP McAb was obtained. The antibody subclass of 1A6, 3C5, 4C4 were IgG2b equally, The antibody subclass of 2D3 was IgG3, light chains were κ chain equally. 4 monoclonal cell supernatant titers were more than 1∶103, ascites titer reaching more than 1∶107. Western blot analysis indicates four monoclonal antibodies can specifically combine with recombinant protein CIRP and had no response with void vector comparison. The ascites of cell line 3C5 was as primitive antibody to test natural CIRP expression status of a variety of cold treated organizations. It shows the ascites of cell line 3C5 can specifically recognize natural CIRP protein of heart, liver, spleen, lung, kidney, brain, testes and other tissues, and can be combined with CIRP McAb. This proves that the CIRP McAb has a good specificity and binding activity. McAb against CIRP will act as biological reagents to be used in study of the further research of CIRP functions and utilization.
|
Received: 29 October 2009
Published: 25 March 2010
|
Corresponding Authors:
shize li
E-mail: lishize1@sina.com
|
|
|
[1] Nishiyama H, Itoh K, Kaneko Y, et al. A glycinerich RNAbinding protein mediating coldinducible suppression of mammalian cell growth. J Cell Biol, 1997, 137(4):899908.
[2] van Venrooy S, Fichtner D, Kunz M. Coldinducible RNA binding protein (CIRP), a novel XTcf3 specific target gene regulates neural development in Xenopus. Bio Med Central, 2008,7(8):77.
[3] Peng Y, Kok K H, Xu R H, et al. Maternal cold inducible RNA binding protein is required for embryonic kidney formation in Xenopus laevis. FEBS Lett, 2000, 482(12)):3743.
[4] Tan H K,Yap M G. Overexpression of coldinducible RNAbinding protein increases interferongamma production in Chinesehamster ovary cells. Biotechnol Appl Biochem, 2008, 49(pt4):247257.
[5] Nishiyama H, Higashitsuji H, Yokoi H, et al. Cloning and characterization of human CIRP(coldinducible RNAbinding protein) cDNA and chromosomal assignment of the gene. Gene, 1997, 204(12):115120.
[6] Nishiyama H, Danno S, Kaneko Y, et al. Decreased expression of coldinducible RNAbinding protein (CIRP) in male germ cells at elevated temperature. Am J Pathol, 1998, 152(1):289296.
[7] Matsumoto K, Aoki K, Dohmae N, et al. CIRP2, a major cytoplasmic RNAbinding protein in Xenopus oocytes. Nucleic Acids Research, 2000, 28(23):46894697.
[8] Yoon S K, Hong J K, Choo S H, et al. Adaptation of Chinese hamster ovary cells to low culture temperature: Cell growth and recombinant protein production. J Biotechnol, 2006, 122(4): 463472.
[9] Nishiyama H, Xue J H, Sato T, et al. Diurnal change of the cold inducible RNAbinding protein (CIRP) expression in mouse brain. Biochem Biophys Res Commun, 1998, 245(2):534538.
[10] Saito T, Sugimoto K, Adachi Y, et al. Cloning and characterization of amphibian cold inducible RNAbinding protein. Com Biochem Physiol B Biochem Mol, 2000, 125(2):237245.
[11] Uochi T, Asashima M. XCIRP(Xenopus homolog of coldinducible RNAbinding protein) is expressed transiently in developing pronephros and neural tissue. Gene, 1998,211(2):245250.
[12] Leeuw F D, Zhang T, Wauquier C, et al. The coldinducible RNAbinding protein migrates from the nucleus to cytoplasmic stress granules by a methylationdependent mechanism and acts as a translational repressor. Exp Cell Res, 2007, 313(20):41304144.
[13] Tanaka K, Kawamura H, Matsugu H. An ascidian glycinerich RNA binding protein is not inducibled by temperature stress but is expressed under a genetic program during embryogenesis. Gene, 2000, 243(12):207214.
[14] Suqimoto K, Jiang H. Cold stress and light signals induce the expression of coldinducible RNA binding protein (cirp) in the brain and eye of the Japanese tree frog. Comp Biochem Physiol A Mol Inteq Physiol, 2008,151(4):324331.
[15] Hamid A A, Mandai M, Fujita J, et al. Expression of coldInducible RNAbinding protein in the normal endometrium, endometrial hyperplasia, and endometrial carcinoma. J Gynecol Pathol, 2003, 22(3):240247.
[16] 李士泽,金福厚,赵巧香,等.冷诱导RNA结合蛋白的生物学功能研究进展.生理科学进展,2009,40(3):271273. Li S Z,Jin F H,Zhao Q X,et al.Chinese Journal of Applied & Environmental Biology,2009,15(1):8790.
[17] 金福厚,庞岩,李士泽,等.BALB/C鼠睾丸组织中冷诱导RNA结合蛋白的cDNA克隆与序列分析.应用与环境生物学报,2009,15(1):8790. Jin F H,Pang Y,Li S Z,et al.Progress in Physiological Sciences,2009,40(3):271273. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
|
Shared |
|
|
|
|
|
Discussed |
|
|
|
|