Research Of The Distribution In Mice Viscera And Expression Rule In IBD Model Of Cryptdin 4

The endogenous antibacterial peptide which extensivly resides in organism is a kind of effective factor with the function of immunity and defense. As it has the characteristic of broad-spectrum anti-pathogenic microorganism and no survivability, so it became to receive people's widespread attention. The major antibacterial peptide in mammal includes defensins, cathelicidins and histstins. Defensins is micromo lecular antibacterial peptide with positive charge, they are effective factors of innate immunity and also regulatory factors of adaptive immunity. There three kind of mammal defensins based on the structural diversity, named asα,βandθ. The cryptdins of mouse is a important kind ofα- defensins.α- defensins is mainly expressd in heterophil granulocyte, macrophagus, Paneth's cell in mammalian small intestine, also expressed in endothelial cell of mammalian alimentary canal and genitourinary tract. Theα- defensins is presumed to have the function of killing microbes, anti- virus, chemical chemotaxis, activating phagocytic cell, inducing cytokine, causing the release of Histamine, restraining adrenal cortex hormone, immune enhancement effect, accommodating complement activity and so on, but the regulative route and mechanism of genetic expression for mammalian antibacterial peptide are indefinite. The purpose of my study is to detect the distribution of cryptdin 4 in mouse and the genetic expression regularity of cryptdin 4 in enteritis model, providing rationale for the study of the mechanism of action of cryptdin 4.The recombinant expression plasmid pMAL-129 is constructed successfully, and a 46kDa protein induced by IPTG is obtained. The protein was proved to have biological activity after purification. The purity coefficient of the protein which was purificated by pMAL? Amylose Pre-packed Column was up to 94%.The protein was used to immune the New Zealand albino rabbit, then collected the whole blood by carotid artery when the valence of antibody reached 14000 by ELISA analysis, the antiserum was obtained. The purity coefficient of antibody purified by octanoic acid-ammonium sulfate method is up to 90%. The pFast-129 calibration protein that expressed in insects cell sf9 was utilized to detect the specificity of purified antibody with western blotting , the result showed that a visible strap was appeared at 8kDa. The ileum, bronchus, spermaductus and oviduct of SVJ/129 were detected by immunohistochemistry, according to the research of the distribution ofα- defensins in the animal. The result showed that the positive result only exist in the Paneth's cell of intestinal gland's basilar part while the other three results were all negative. The result indicated that the cryp4 could be expressed in ileum. The distribution of cryp 4 can depart the digestive system of small intestine and its anterior segment from posterior large intestine, meanwhile, it can protect the balance of flora in the intestine, because it can restrain or kill bacterium and virus.After dabbled colons of mice with TNBS/alcohol mixture to induce inflamed Enteritis, the intestinal mucosa desquamated and the activity of myeloperoxidase rised, which demonstrated that patterns were established successfully. Two pairs of specific primers were designed according to cryp4 and GAPDH of mice which were published in GeneBank. The exprission of cryp4 in ileum of mice was detected by fluorescent quantitation PCR .The results indicated that the designed primers have good specificity, so PCR detection with SYBR GREENⅠwas carried out .The results which were analyzed by ABI 7000 System SDS Software demonstrated that the quantity of cryp4 reached the maximum at 24h after dabbling, and then begin to decreased, reaching the minimum on the fourth day. Then it rised a little, but still less than normal level. The possible reasons were as follows: cryp4 acted as the endogenous immune factor at the primary stage, and the quantity raised. However, with the development of the inflammation, the quantity decreased because the ileal mucous membrane was destroyed. It was also possible that the quantity decrease of cryp4 is the result of synergistic effect of the immunoregulation and physical damage.The conclusions of the experiment provided substantial rationale for the further study of the cryptdin 4.