Production And Application Of Polyclonal Antibody Of Mouse IFIT1

PART ONE:PRODUCTION OF POLYCLONAL ANTIBODY OF MOUSE IFIT1Objective: To acquire specific polyclonal antibody of mouse IFIT1 for the sake of deep investigation of its structure and function.Methods: The single clone of pMAL-C2X-IFIT1, which was confirmed previously to highly express MBP-IFIT1 fusion protein, was cultivated and amplified. After ultrasonic fragmentation and centrifugation, the MBP-IFIT1 fusion protein was purified from the cellular supernatant by passing through the Amylose Pre-packed column. Then, the MBP-IFIT1 fusion protein mixed with Freund,s Adjuvant was injected into rabbits twice with two weeks interval. The serum was collected, then its anti-IFIT1 titer was detected by ELISA, and its anti-IFIT1 specific reaction was detected by Western blot .Results: The serum collected from the rabbits immuned with purified MBP-IFIT1 protein showed specific anti- IFIT1 ability, and its antibody titer was about 1:12800 estimated by ELISA.Conclusion: Purified MBP-IFIT1 fusion protein possesses favorable antigenicity, and highly specific ployclonal antibody of mouse IFIT1 can be obtained by using purified MBP-IFIT1 as antigen to immune rabbit.PART TWO:THE EXPRESSION OF IFIT1 IN VARIOUS ORGAN OF MICE AND CELLSObjective: To study the expression of IFIT1(Interferon-induced protein with tetratricopetide repeats 1)in various organ of mice and cells.Methods: RNA was extracted from the heart, liver, spleen, lung, kidney, small intestine, lymphocyte and skeletal muscle of normal male C57/129 mice, and RNA was extracted from Raw264.7 cell, 3T3 cell and 10T1/2 cell after 5h stimulated with 5μg/ml LPS, at the same time, to set up normal control group(untreated by LPS). Then the RNA expression level of IFIT1 was detected by RT-PCR.Results: the RNA expression level of IFIT1 was detected in the heart, liver, spleen, lung, kidney, small intestine, lymphocyte and skeletal muscle of normal male, but there is difference in various organ, the highest is heart, liver compared with small intestine p=0.307,the other tissues compared p<0.05. After 5h stimulated with LPS, IFIT1 was induced expression in Raw264.7 cell, 3T3 cell and 10T1/2 cell.Conclusion: These results suggest that IFIT1 is generally expressed in various organ of mice and LPS can stimulated multi-cell expressed IFIT1.PART THREE:REFLECTION OF THE HEPATIC IFIT1 AND JAB1 - ASSOCIATED PROTEIN OF IRRITABILITY DURING THE EARLY STAGE IN RADATION INJURY MICEObjective:To observe the characteristic of the hepatic IFIT1 and JAB1 during the early stage every phase in radiation injury mice, to discuss the molecule mechanism of glucocorticoid resistance in the early stage of wounded.Methods: twenty C57 mice were randomly divided into control group and vulnerate group. The mice in vulnerate group were given 5Gry 60Co full-body exposure once. IFIT1 and JAB1 were respectively detected by western blot at 1, 4 and 12 hours after radiation injury.Results: One hour after radiation injury, the levels of IFIT1 were increased significantly, and reached in the peak at twelve hours(P<0.01); the levels of JAB1 were decreased significantly, and reached the lowest at twelve hours (P<0.01).Conclusion : The levels of IFIT1 were increased significantly and the levels of JAB1 were decreased significantly during the early stage in radiation injury mice. To reveal glucocorticoid resistance relative to repression of IFIT1 and up-regulation of JAB1 for it in the early stage of wounded.PART FOUR: EFFECT OF CERVICAL SYMPATHETIC BLOCK TO THE INTERFERON-INDUCED PROTEIN IFIT1 IN HEPATIC OF BURNED MICE DURING THE EARLY STAGEObjective: To detect the effect of cervical sympathetic block(SB) to IFIT1 (Interferon-induced protein with tetratricopetide repeats 1)of hepatic during the early stage in burned mice.Methods: Fifty male C57/129 mice inflicted with 15-20% TBSA full thickness burn injury were divided randomly into control group (untreated by SB) and SB group(treated immediately by SB). Hepatic tissue was taken before burn injury and at 1 h,6 h,12 h and 24 h after burn injury. The protein expression level of IFIT1 were measured by Western blot.Results: The result of Western blot display that the protein expression level of IFIT1 in control group increased significantly(P<0.01). The level of IFIT1 in SB group was decreased significantly at 1h, 6h,12h and 24h after burn injury compared with the control group(P<0.01).Conclusion: These results suggest that the supression of GR after burn injury is concerned with negativity regulation of IFIT1. SB could enhance function of GR by exerting supression on the level of IFIT1.