重组鸡白细胞介素18生物学活性检测及其对鸡免疫影响的研究
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摘要
白细胞介素18(IL-18)具有广泛的生物学活性,能够促进γ-干扰素(IFN-γ)的产生,刺激淋巴细胞转化,增强NK细胞的杀伤活性,在介导细胞免疫、抵抗微生物感染方面具有重要的作用。对人和哺乳动物IL-18的研究已有许多报道。本研究室在2001年开展鸡白细胞介素18(ChIL-18)的研究,先后构建ChIL-18的重组质粒和原核表达系统、真核质粒及其表达系统,在此基础上,对纯化后的原核表达的重组鸡白细胞介素18产物进行生物学活性检测,对其在生产中的应用做了初步研究,同时对影响ChIL-18原核表达产物生物学活性的因素也进行了探讨。
实验一MTT法检测鸡脾和外周血NK细胞杀伤活性方法的建立
选取不同日龄的SPF鸡和肉鸡,通过贴壁法从血液和脾脏分离出NK细胞作为效应细胞,选择MDCC-MSB1和MDCC-MSBCU147分别作为靶细胞,二者按比例混合,孵育,利用MTT法检测靶细胞的活力反映效应细胞对靶细胞的杀伤活性,并对效靶比、作用时间进行比较,结果显示MSB-1和CU147均可作为NK细胞良好的靶细胞,但CU147更稳定;效靶比为20:1~50:1,作用时间8~16h杀伤效果稳定;50%DMF- 20%SDS混合物作为裂解液能够对结晶彻底溶解,优于其它溶解试剂如二甲基亚砜等。该方法为检测重组鸡白细胞介素18蛋白对NK细胞杀伤作用这一生物学活性奠定基础。
实验二鸡白细胞介素-18(ChIL-18)重组蛋白的生物学活性检测
对含有ChIL-18基因的质粒在大肠杆菌内进行诱导表达,经亲合层析法对表达产物进行纯化,用微量细胞病变抑制法——CEF-VSV系统,检测其对SPF鸡脾淋巴细胞诱导产生γ-干扰素(IFN-γ)的活性和其对病毒的抑制效果;用3H-TdR掺入法和MTT法分别检测其对T淋巴细胞诱导转化和NK细胞杀伤活性的作用。结果表明经诱导表达出分子量为44KD的目的蛋白(与GST融合表达),纯化后得到去除菌体蛋白的高纯度目的蛋白;生物学活性检测表明该蛋白能够诱导脾细胞产生IFN-γ,当浓度为250ng/ml时诱导IFN-γ的活性最高,可达1×106U/ml;但该蛋白不能直接抑制VSV在CEF上生长;能够刺激淋巴细胞显著增殖,浓度为250ng/ml时效果最佳;适当浓度的rChIL-18蛋白能促进NK细胞的杀伤活性,浓度为150ng/ml时,作用最强。以上结果表明,利用原核表达的重组鸡白细胞介素-18蛋白具有与天然鸡IL-18蛋白一样较广泛的生物学活性,为进一步研究它在生产中的应用奠定了基础。
Interleukin 18(IL-18) has multiple biological activities including induction of IFN-γfrom NK cells and antigen- or mitogen-stimulated Th1 cells, upregulation of IL-2R on T cells, enhancement of Fas ligand-mediated cytotoxicity of T-helper cells and augmentation of NK cell cytotoxicity. These on human beings and animals were identified by many documents recently. Chickens IL-18 was constructed to recombinant chicken IL-18 genes in our previous study in 2003. The bioactivities of expressed product of ChIL-18 in E.coli would be assayed and the methods of purification and renaturation will be assessed and the effect of it on the immune in chickens will be study as following.
1 Optimal Conditions in the MTT Assays to Detect the Cytotoxicity of NK Cells
The cytotoxicity of NK cells isolated from blood and spleen of different-day-chickens as effect cells to target cells, MDCC-MSB1 and MSBCU147, were detected by MTT assays and evalue the ratio of effect cells and target cells and the time of incubation. The result indicated MSB1 and MSBCU147 could be better target cells in the assay of NK cells cytotoxicity and the optimal conditions were 20:1~50:1 as the ratio of effect cell and target cells and 8~16h of incubation and the 20%SDS-50%DMF mixture as lytsis to MTT product. This will be help to analyze the cytotoxicity of NK cells in chickens.
2 Biological Activities Assay of Purified Expressed Product of Recombined Chicken Interleukin-18 Gene in E.coli
Positive vector containing chicken IL-18 gene was expressed in E.coli, then the expressed product was purified by affinity chromatography, the activity of IFN-γinduced by rChIL-18 and of rChIL-18 antivirus were detected with CEF-VSV system, 3H-thymidine (3H-TdR) incorporation assay and MTT colorimetric assay were used to detect T lymphocytes proliferation response to rChIL-18 and the effect of rChIL-18 on cytotoxicity of NK cells, respectively. Results, recombinant protein with a relative molecular weight of 44 KD (including 26KD GST) was expressed in E.coli, which could induce splenocytes to produce IFN-γwith high activity, but could not directly inhibit the growth of VSV in CEF. It was proved that purified protein could stimulate T lymphocytes proliferation and increase NK cytotoxicity at certain concentrations. It can be concluded that chicken IL-18 protein
实验一MTT法检测鸡脾和外周血NK细胞杀伤活性方法的建立
选取不同日龄的SPF鸡和肉鸡,通过贴壁法从血液和脾脏分离出NK细胞作为效应细胞,选择MDCC-MSB1和MDCC-MSBCU147分别作为靶细胞,二者按比例混合,孵育,利用MTT法检测靶细胞的活力反映效应细胞对靶细胞的杀伤活性,并对效靶比、作用时间进行比较,结果显示MSB-1和CU147均可作为NK细胞良好的靶细胞,但CU147更稳定;效靶比为20:1~50:1,作用时间8~16h杀伤效果稳定;50%DMF- 20%SDS混合物作为裂解液能够对结晶彻底溶解,优于其它溶解试剂如二甲基亚砜等。该方法为检测重组鸡白细胞介素18蛋白对NK细胞杀伤作用这一生物学活性奠定基础。
实验二鸡白细胞介素-18(ChIL-18)重组蛋白的生物学活性检测
对含有ChIL-18基因的质粒在大肠杆菌内进行诱导表达,经亲合层析法对表达产物进行纯化,用微量细胞病变抑制法——CEF-VSV系统,检测其对SPF鸡脾淋巴细胞诱导产生γ-干扰素(IFN-γ)的活性和其对病毒的抑制效果;用3H-TdR掺入法和MTT法分别检测其对T淋巴细胞诱导转化和NK细胞杀伤活性的作用。结果表明经诱导表达出分子量为44KD的目的蛋白(与GST融合表达),纯化后得到去除菌体蛋白的高纯度目的蛋白;生物学活性检测表明该蛋白能够诱导脾细胞产生IFN-γ,当浓度为250ng/ml时诱导IFN-γ的活性最高,可达1×106U/ml;但该蛋白不能直接抑制VSV在CEF上生长;能够刺激淋巴细胞显著增殖,浓度为250ng/ml时效果最佳;适当浓度的rChIL-18蛋白能促进NK细胞的杀伤活性,浓度为150ng/ml时,作用最强。以上结果表明,利用原核表达的重组鸡白细胞介素-18蛋白具有与天然鸡IL-18蛋白一样较广泛的生物学活性,为进一步研究它在生产中的应用奠定了基础。
Interleukin 18(IL-18) has multiple biological activities including induction of IFN-γfrom NK cells and antigen- or mitogen-stimulated Th1 cells, upregulation of IL-2R on T cells, enhancement of Fas ligand-mediated cytotoxicity of T-helper cells and augmentation of NK cell cytotoxicity. These on human beings and animals were identified by many documents recently. Chickens IL-18 was constructed to recombinant chicken IL-18 genes in our previous study in 2003. The bioactivities of expressed product of ChIL-18 in E.coli would be assayed and the methods of purification and renaturation will be assessed and the effect of it on the immune in chickens will be study as following.
1 Optimal Conditions in the MTT Assays to Detect the Cytotoxicity of NK Cells
The cytotoxicity of NK cells isolated from blood and spleen of different-day-chickens as effect cells to target cells, MDCC-MSB1 and MSBCU147, were detected by MTT assays and evalue the ratio of effect cells and target cells and the time of incubation. The result indicated MSB1 and MSBCU147 could be better target cells in the assay of NK cells cytotoxicity and the optimal conditions were 20:1~50:1 as the ratio of effect cell and target cells and 8~16h of incubation and the 20%SDS-50%DMF mixture as lytsis to MTT product. This will be help to analyze the cytotoxicity of NK cells in chickens.
2 Biological Activities Assay of Purified Expressed Product of Recombined Chicken Interleukin-18 Gene in E.coli
Positive vector containing chicken IL-18 gene was expressed in E.coli, then the expressed product was purified by affinity chromatography, the activity of IFN-γinduced by rChIL-18 and of rChIL-18 antivirus were detected with CEF-VSV system, 3H-thymidine (3H-TdR) incorporation assay and MTT colorimetric assay were used to detect T lymphocytes proliferation response to rChIL-18 and the effect of rChIL-18 on cytotoxicity of NK cells, respectively. Results, recombinant protein with a relative molecular weight of 44 KD (including 26KD GST) was expressed in E.coli, which could induce splenocytes to produce IFN-γwith high activity, but could not directly inhibit the growth of VSV in CEF. It was proved that purified protein could stimulate T lymphocytes proliferation and increase NK cytotoxicity at certain concentrations. It can be concluded that chicken IL-18 protein
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