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대장균에서 재조합 인간 Interleukin 4 의 생산 , 정제 및 면역조절활성의 측정
양영,윤석란,이충은,변광호 ( Young Yang,Suk Ran Yoon,Choong Eun Lee,Kwang Ho Pyun ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.1
The recombinant human interleukin 4 (rhIL-4) has been over expressed in E. coli transformed with expression vector pET-3b containing bacteriophage T7 promoter, into which the hIL-4 cDNA was subclond. The insolubility of the recombinant protein offered an advantage of purification in only a few steps. The recombinant human IL-4 was refolded using reduced/oxidized glutathione to restore the proper conformation and purified to homogeneity by one passage over ion exchange column. The purified protein was shown as a single band on SDS-PAGE. The refolded rhIL-4 was characterized by nucleotide sequence analysis and bioassays. The purified rhIL-4 has biological activities on B cell proliferation and induction of B cell differentiation antigen, CD23, which strongly indicates that the protein is folded correctly.
대장균에서 재조합 인간 Interleukin 6의 대량생산 및 그 생물학적 활성의 측정
양영,강형식,나우진,이충은,변광호 ( Young Yang,Hyung Sik Kang,Woo Jin Na,Choong Eun Lee,Kwang Ho Pyun ) 생화학분자생물학회 1992 BMB Reports Vol.25 No.2
Human interleukin 6 (hIL-6) is a multifunctional cytokine involved in acute phase and immune response. While a critical role of IL-6 in various immune disorders has been suggested, studies on its detailed functional mechanism are often hampered due to the low natural abundance of this cytokine. Thus, for a large scale production of hIL-6, we have made an attempt to directly express hIL-6 using pET-8c expression plasmid under the control of T7 promoter in Escherichia coli. A cDNA coding for hIL-6 without the signal sequence was amplified using PCR reaction, fused to pET-8c, and transformed into E. coli (λDE3). Upon induction with isopropyl thiogalactoside, recombinant human interleukin 6 (rhIL-6) was overexpressed as a form of inculsion bodies, with its expression level reaching over 30% of total E. coli proteins. The rhIL-6 was isolated from inclusion bodies by solubilization in 6 M guanidine hydrochloride followed by dialysis against 50 mM Tris buffer. A single passage over DEAE-Sepharose faciliated the purification of rhIL-6. The purified rhIL-6 was characterized by nucleotide sequence analysis and bioassays. The biological activity was confirmed by proliferation of B9 cells and immunoglobulin secretion of SAC-blast B cells.
김성숙(Sung Sook Kim),김도영(Doe Young Kim),문일환(Il Whan Moon),변광호(Kwang Ho Pyun),최인표(In Pyo Choi) 대한소화기학회 1995 대한소화기학회지 Vol.27 No.4
N/A Rackground/Aims: Interleukin-6 (lL-6), also known as B cell stimulatory factor 2(BSF-2), induces the final maturation of B cells to antitxxiy-producing cells. IL-6 has many biologic properties including the immune and intlammatory responses. This study wos aimed to evaluate the role of local interleukin 6(IL-6) in the pathogenesis of chronic hepatitis. Methods; We examined the cellular site and grade of IL-6 staining in paraffin sections of the liver from 24 patients with liver disease, using immunohistochemistry with a polyclonal antitwdy. The patient. Were divided into two groups; Group A(n=l3) with high histologic uctivi1y consisted of CAH-type B(n=10) ond active cirrhosis(n=3), whilc Group B(n= l l) with low hi.itologic activity consisted of CPH-type B(n=4), inactive cirrhosis(n=2) and fatty liver(n=S). Results: There was no staining of IL-6 in normal liver tissue. Thv grade.I of IL-6 staining in Group A were three positive in seven cases (53.81o), two positive in five ca.ics(38.3%) and one positive in only one case(7.7%), while those in Group B were one positivc in three cases(27.3%) ancl trace in eight case.(72.7ln). IL-6 stained cells in chronic hepatitis were hepatocytcs, cspecially in the areu ot' piecemeol necrosi.I, bilc duct cel1., infiltrating inflammatory cells and endothelial cell.I. The score of histological activity index(HAJ), piecemeal necrosis and fibrasis and thc gradv. Of 1L-6 staining of Group A were ull significantly higher than those of Group B. The grade of IL-6 staining and HAI werc well correlated(r =0.74, p 0.0l), Conclusion: Locally produced IL-6 in the liver may contribute to the inflammatory process and immunological response in chronic hepatiti.. (Korean 3 Gastroenterol 1995;27:403-411)
Interleukin 4가 각질형성세포로부터의 Interleukin 6 생산에 미치는 영향
조상현 ( Sang Hyun Cho ),김진우 ( Jin Wou Kim ),변광호 ( Kwang Ho Pyun ),김정원 ( Chung Won Kim ),허원 ( Won Houh ) 대한피부과학회 1995 大韓皮膚科學會誌 Vol.33 No.5
Background : The hum;n keratinocyte can synthesize interleukin 6 (IL-6) under certain concitions, and the IL-6 syntiesis is inhibited by interleukin 4 (IL-4) in the human monocyte. Objective : To find ou, what kind of stimulating agents can induce the IL-6 production and whether IL-4 affects the production of IL 6 in the human cultured keratinocytes. Methods : We stimulated the keratinocytes with either lipopolysaccharide (LPS), fetal bovine serum (FBS), human re ombinant interferon-r(IFN-r) to induce the IL-6 production, and treeted the keratinocytes, wh:ch stimulated either with 10% FBS or human recombinant IFN-r, with human recombinant IL-4. Results : The LPS stimulation resulted in no increase of IL-6 levels in the keratinocyie supernatants. When the keratinocytes were stimulated either with 1%, 5%, 10% FBS with or without 5 pg/ml LPS, s gnificantly increased amounts of IL 6 were detected. The level of IL 6 in the keratinocytes treated with the human recombinant IFN-r increased, too. The human recornbinant IL-4 downregulats the secretion of IL-6 by the keratinocytes which were activated eithr with 10% FBS or human recombinant, IFN-r. Conclusion : We have shown that it is possible to induce the IL-6 synthesis by stimulating the keratinocytes and that, human recombinant IL-4 profoundly inhibits the synthesis of IL-6. So we suggest, that there may be a cytokine network which regulates the pr imary immune response in the skin. (Kor J Dermatol 1995;33(5): 847-854)