鸡白细胞介素2在毕赤酵母中的表达与生物活性分析
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摘要
家禽传染性疾病是养禽业中最大的威胁,给养殖业造成严重经济损失,免疫预防则是控制疾病的重要战略。但是在实际生产中,并不是所有疾病均有疫苗可用,也不是所有疫苗均可对机体产生完全免疫保护。特别是一些灭活苗和基因工程苗由于抗原性低等原因,免疫效果往往并不理想,时常造成免疫失败。
鸡白细胞介素-2(Chicken Interleukin-2, ChIL-2)在家禽养殖业中可作为免疫佐剂,提高鸡体抵抗病原的免疫力,自1982年被发现以来一直是细胞因子研究中的热点之一,可望与疫苗配合使用,成为一种重要的细胞免疫佐剂,提高机体抗感染力,增强灭活苗或基因工程疫苗免疫效果。
本文利用毕赤酵母(Pichia Pastoris)表达重组ChIL-2并对其提高鸡免疫功能的生理活性作了研究。首先将ChIL-2克隆至毕赤酵母穿梭表达质粒pPIC9K构建重组质粒pPIC9K/ChIL-2,经Sal I线性化酶切,电穿孔将其整合到毕赤酵母GS115(His+Mut+)染色体上,利用MM、MD平板筛选His+Mut+型阳性克隆,再用高浓度G418-YPD平板筛选高拷贝转化子。共筛选到了5株阳性克隆,用菌落PCR做进一步的筛选,得到3株高拷贝菌株。筛选到的高拷贝菌株进行BMGY/BMMY摇瓶培养,诱导表达重组ChIL-2。经SDS-PAGE与Western blot分析,证实ChIL-2基因在重组GS115中成功表达出约16 kDa与14 kDa两条特异性条带,这与天然ChIL-2分子量大小一致,提示与天然蛋白一样,重组ChIL-2可能也为糖基化蛋白。
将重组的酵母菌株进行发酵生产,其表达量较摇瓶培养提高了20倍左右,达到约100 mg/L,重组目标蛋白质量分数达总蛋白的50 %以上。由于目的蛋白与杂蛋白带大小相差甚大,通过膜过滤截留就得到了高纯度的目的蛋白,操作简单。本研究为工业化生产鸡白细胞介素-2奠定了基础。
将纯化到的ChIL-2与疫苗一起联合免疫鸡群,研究其作为免疫增强剂对禽流感病毒(avian influenza virus, AIV)疫苗和新城疫病毒(Newcastle disease virus, NDV)疫苗的免疫增强效果,用血凝抑制试验检测鸡体血清中的AI抗体和ND抗体水平。实验结果表明:ChIL-2与禽流感或新城疫疫苗同时使用对疫苗有良好的免疫增强作用,能有效提高鸡体抗体滴度,延长抗体滴度的时间,提高存活率。ChIL-2的使用剂量应该在8.35μg/鸡左右。在使用的过程中,ChIL-2没有发现任何毒副作用,证明是一种安全高效的免疫增强剂。
Domestic infective diseases wich caused the tremendous economic losses to poultry industry, were the most large threat of poultry industry. The application of vaccines was the most improtant strategy to guard against these diseases. In the poultry industry, there are various vaccines to different diseases, but they are not effective to all diseases. In addition, vaccines could not entirely protect fowls from given disease. In particular, the low antigenicity of inactivated vaccines and genetically engineered vaccines often cause failure during immunization.
Chicken interleukin-2 (ChIL-2) can be used as immunological adjuvant in the poultry industry to enhance the immunity of chickens against pathogens. It had been a focus in the cytokines’study since it was found in 1982. It was used as an important adjuvant in cell immunity.
In the present study, the chicken interleukin-2 gene was inserted into the expression vector pPIC9K to construct recombinant plasmid (pPIC9K/ChIL-2) followed by linearizing by restriction endonuclease Sal I. Then it was transformed into host Pichia Pastoris GS115 by eletroporation. After screening by G418-YPD, following by MM and MD plates, we obtained 5 positve clonies (His+Mut+). In the next PCR, 3 clonies with high-copy of purpose gene were obtained. Among them, one colony was cultured in BMGY/BMMY flask for the expression of ChIL-2 in vitro. The expression of recombinant ChIL-2 was successfully performed. Similarly to native ChIL-2, the expressed ChIL-2 revealed two main bands corresponding to molecular masses of 16 and 14 kDa, respectively as detected by SDS-PAGE and Western blot, suggesting it could be a glycosylated protein.
By fermentation, the production of recombinant protein increased about 20-fold, up to 100 mg/L. In the total cell proteins, the target protein ChIL-2 occupied more than 50 %. Because the great difference of molecular masses between the proteins in the cell culture, membrane ultrafiltration purification was applied to ChIL-2 purification and was a convenient way. The present study established a foundation for the industrial production of ChIL-2.
Purified ChIL-2 was co-admitted with avian influenza virus (AI) vaccine and newcastle disease (ND) vaccine to study its immunological enhancing effect on AI and ND in chickens.The antibody titers of AI and ND were detected by hemagglutination inhibition test. The results indicated that ChIL-2 had positive immuno-enhancing effect on both vaccines. The immuno-enhancing effects were respresented by antibody titer increasing and its extending. ChIL-2 also increased the survival rate of the chickens. The optimum dosage of ChIL-2 as adjuvant was about 8.35μg/chicken.During the whole experiment periods,no toxic effect of ChIL-2 was identified, suggesting it was an effective and safe adjuvant for vaccination.
鸡白细胞介素-2(Chicken Interleukin-2, ChIL-2)在家禽养殖业中可作为免疫佐剂,提高鸡体抵抗病原的免疫力,自1982年被发现以来一直是细胞因子研究中的热点之一,可望与疫苗配合使用,成为一种重要的细胞免疫佐剂,提高机体抗感染力,增强灭活苗或基因工程疫苗免疫效果。
本文利用毕赤酵母(Pichia Pastoris)表达重组ChIL-2并对其提高鸡免疫功能的生理活性作了研究。首先将ChIL-2克隆至毕赤酵母穿梭表达质粒pPIC9K构建重组质粒pPIC9K/ChIL-2,经Sal I线性化酶切,电穿孔将其整合到毕赤酵母GS115(His+Mut+)染色体上,利用MM、MD平板筛选His+Mut+型阳性克隆,再用高浓度G418-YPD平板筛选高拷贝转化子。共筛选到了5株阳性克隆,用菌落PCR做进一步的筛选,得到3株高拷贝菌株。筛选到的高拷贝菌株进行BMGY/BMMY摇瓶培养,诱导表达重组ChIL-2。经SDS-PAGE与Western blot分析,证实ChIL-2基因在重组GS115中成功表达出约16 kDa与14 kDa两条特异性条带,这与天然ChIL-2分子量大小一致,提示与天然蛋白一样,重组ChIL-2可能也为糖基化蛋白。
将重组的酵母菌株进行发酵生产,其表达量较摇瓶培养提高了20倍左右,达到约100 mg/L,重组目标蛋白质量分数达总蛋白的50 %以上。由于目的蛋白与杂蛋白带大小相差甚大,通过膜过滤截留就得到了高纯度的目的蛋白,操作简单。本研究为工业化生产鸡白细胞介素-2奠定了基础。
将纯化到的ChIL-2与疫苗一起联合免疫鸡群,研究其作为免疫增强剂对禽流感病毒(avian influenza virus, AIV)疫苗和新城疫病毒(Newcastle disease virus, NDV)疫苗的免疫增强效果,用血凝抑制试验检测鸡体血清中的AI抗体和ND抗体水平。实验结果表明:ChIL-2与禽流感或新城疫疫苗同时使用对疫苗有良好的免疫增强作用,能有效提高鸡体抗体滴度,延长抗体滴度的时间,提高存活率。ChIL-2的使用剂量应该在8.35μg/鸡左右。在使用的过程中,ChIL-2没有发现任何毒副作用,证明是一种安全高效的免疫增强剂。
Domestic infective diseases wich caused the tremendous economic losses to poultry industry, were the most large threat of poultry industry. The application of vaccines was the most improtant strategy to guard against these diseases. In the poultry industry, there are various vaccines to different diseases, but they are not effective to all diseases. In addition, vaccines could not entirely protect fowls from given disease. In particular, the low antigenicity of inactivated vaccines and genetically engineered vaccines often cause failure during immunization.
Chicken interleukin-2 (ChIL-2) can be used as immunological adjuvant in the poultry industry to enhance the immunity of chickens against pathogens. It had been a focus in the cytokines’study since it was found in 1982. It was used as an important adjuvant in cell immunity.
In the present study, the chicken interleukin-2 gene was inserted into the expression vector pPIC9K to construct recombinant plasmid (pPIC9K/ChIL-2) followed by linearizing by restriction endonuclease Sal I. Then it was transformed into host Pichia Pastoris GS115 by eletroporation. After screening by G418-YPD, following by MM and MD plates, we obtained 5 positve clonies (His+Mut+). In the next PCR, 3 clonies with high-copy of purpose gene were obtained. Among them, one colony was cultured in BMGY/BMMY flask for the expression of ChIL-2 in vitro. The expression of recombinant ChIL-2 was successfully performed. Similarly to native ChIL-2, the expressed ChIL-2 revealed two main bands corresponding to molecular masses of 16 and 14 kDa, respectively as detected by SDS-PAGE and Western blot, suggesting it could be a glycosylated protein.
By fermentation, the production of recombinant protein increased about 20-fold, up to 100 mg/L. In the total cell proteins, the target protein ChIL-2 occupied more than 50 %. Because the great difference of molecular masses between the proteins in the cell culture, membrane ultrafiltration purification was applied to ChIL-2 purification and was a convenient way. The present study established a foundation for the industrial production of ChIL-2.
Purified ChIL-2 was co-admitted with avian influenza virus (AI) vaccine and newcastle disease (ND) vaccine to study its immunological enhancing effect on AI and ND in chickens.The antibody titers of AI and ND were detected by hemagglutination inhibition test. The results indicated that ChIL-2 had positive immuno-enhancing effect on both vaccines. The immuno-enhancing effects were respresented by antibody titer increasing and its extending. ChIL-2 also increased the survival rate of the chickens. The optimum dosage of ChIL-2 as adjuvant was about 8.35μg/chicken.During the whole experiment periods,no toxic effect of ChIL-2 was identified, suggesting it was an effective and safe adjuvant for vaccination.
引文
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