clone, Expression, Purification and Characterization of Human Interleukin 4 Alternative Splicing Variant(IL-4δ2)
|School||Fourth Military Medical University|
|Keywords||Interleukin-4 Alternative splicing Polymerase chain reaction Clone Fusion expression Protein purification Asthma|
Bronchial asthma is a multifactorial caused by reversible airway obstruction, airway hyperresponsiveness and airway inflammation characterized by immune allergic diseases, in which airway inflammation is the main pathological changes and decide airway obstruction and airway hyperresponsiveness level. At home and abroad in recent years, researchers at the cellular level, the molecular level and the genetic level in the pathogenesis of asthma conducted in-depth research and found that the incidence of asthma and many other immune cells, numerous cytokines, a variety of biological agents as well as genetic factors. IL-4 is the major regulator of the immune system factors, including its role in induction of T cells to Th2 phenotype, such as activated B cells IgE synthesis. Recent studies indicate that the pathogenesis of allergic asthma without the important role of Th2 cells, which IL-4 Habitat center. Therefore, IL-4 as a new target for drug therapy for the clinical treatment of allergic asthma has opened up a new way of thinking. Study, IL-4 IL-4δ2 optional splice variants, the variant exon 2 is cut off, the exon 1 and exon 3 directly with stitching, to produce only the exon 3 and 4, with a single open reading frame protein. Moreover, IL-4δ2 can act as a natural antagonist of IL-4, by competitive inhibition of IL-4 binding to its receptor and exert its inhibitory effect. In this experiment, we first observed the IL-4δ2 expression in patients with asthma, the results show that, IL-4δ2 in asthma patients and healthy populations were significantly different expression, the former IL-4δ2 expression was significantly higher than the latter. On this basis, we used RT-PCR reaction technology hIL-4δ2 gene was successfully cloned, and its transformation, and was highly expressed in E. coli, expression accounted Army four hood sitting Sushi public position papers Shen Wen bacteria from playing 30% of the total body protein. The main building of the washed inclusion bodies, renaturation, atmospheric sticks of anion and cation exchange chromatography separation and extraction from the E. coli expression product of the production process, the success of purified fusion protein h Bu 4 anus. In order to reduce production costs and simple post-treatment, the introduction of hydroxylamine cleavage site, the proposed means to obtain pure chemical cleavage. Vitro experiments showed, IL 62 fusion protein called several -4 promote T cell proliferation significantly inhibited, and it can significantly inhibit the B cell synthesis and 023 k expression.