Leila Barzegar Yarmohammadi, Mohammad Hossein Modarresi, Saeed Talebi, Reza Hadavi, Mahyar Ostad Karampour, Reza Mahmoudi Ahmad, Mehdi Akhondi Mohammad, Hodjattallah Rabbani, Mahmood Jeddi-Tehrani
Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran ; Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran.
J Reprod Infertil. 2009 Oct;10(3):193-8.
Production of antibodies against specific proteins of testis germ cells is of great significance for the investigation of processes involved in spermatogenesis, study of infertility problems and determination of the probable role of these proteins as cancer-testis antigens. Murine Testis Specific Recombinant Protein 101 (mTEX101) is a 38kDa, GPI-anchored protein which is expressed in testis germ cells of adult mice but it seems to be absent in other tissues. The structure and function of mTEX101 is not completely understood yet, but it is speculated that it may transduce biochemical signals into the cytoplasm since mTEX101 does not have an intracellular domain but the precise mechanisms are still ambiguous.
RNA was extracted from three adult mice testis. The RNA was used in RT-PCR, employing a pair of specific primers for mTEX101 ORF region. TA-cloning technique was performed by the insertion of mTEX101 into a pGEM-T Easy Vector, followed by its subcloning into a His-tagged expression vector, pET-28a (+). The recombinant mTEX101 was then produced by transfection of the expression vector into BL 21 (DE3) E. coli strain.
A recombinant protein, weighing 27kDa, was produced upon IPTG-induction of the bacterial host. The presence of mTEX101 protein was detected through Western blot analysis by anti-mTEX101 peptide antibodies.
We produced mTEX101 recombinant protein that could be used for the production of mono and polyclonal antibodies.
针对睾丸生殖细胞特异性蛋白质产生抗体，对于研究精子发生过程、不育问题以及确定这些蛋白质作为癌-睾丸抗原的可能作用具有重要意义。小鼠睾丸特异性重组蛋白101（mTEX101）是一种38kDa的糖基磷脂酰肌醇锚定蛋白，在成年小鼠的睾丸生殖细胞中表达，但在其他组织中似乎不存在。mTEX101的结构和功能尚未完全明确，但据推测它可能将生化信号转导至细胞质中，因为mTEX101没有细胞内结构域，但其确切机制仍不明确。
从三只成年小鼠的睾丸中提取RNA。该RNA用于RT-PCR，采用一对针对mTEX101开放阅读框区域的特异性引物。通过将mTEX101插入pGEM-T Easy载体进行TA克隆技术，随后将其亚克隆到带有His标签的表达载体pET-28a(+)中。然后通过将表达载体转染到BL 21(DE3)大肠杆菌菌株中来产生重组mTEX101。
在IPTG诱导细菌宿主后产生了一种重27kDa的重组蛋白。通过抗mTEX101肽抗体的蛋白质免疫印迹分析检测到mTEX101蛋白的存在。
我们制备了可用于生产单克隆和多克隆抗体的mTEX101重组蛋白。
