猪β干扰素基因的修饰及其蛋白活性的检测  

Gene modification of porcine interferon beta and its protein activity

作　　者：徐蕙[1] 朱艳平[1] 刘京军[1] 刘德辉[1] 郭霄峰[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《中国兽医学报》2010年第11期1511-1516,共6页Chinese Journal of Veterinary Science

基　　金：2007年广东省动物防疫检疫研究专项经费资助项目(粤财农[2007]479号)

摘　　要：为获得高效表达的重组猪β干扰素,在保留编码蛋白序列的同时,对PoIFN-β基因进行毕赤酵母偏嗜性改造,使基因序列G+C的含量增加到最大限度,并将其终止密码子改为TAA。构建了酵母表达载体pPICZαC-PIB。pPICZαC-PIB经SacⅠ酶切线性化后电转导入毕赤酵母菌株X-33。对PCR鉴定为阳性的酵母菌株进行高抗性筛选,获得1株高效表达PoIFN-β酵母菌株,其表达量约258.3 mg/L,是未经密码子改造菌株表达产物的3.4倍。经SDS-PAGE和Western blotting检测,表达产物为相对分子质量约22 000和24 000的蛋白,两者均可与抗PoIFN-β阳性血清发生特异反应。将表达产物进行氨基酸测序,测得2段随机肽段与PoIFN-β的氨基酸序列完全一致。在VSV-Vero系统上检测重组PoIFN-β对水泡性口炎病毒在Vero细胞中增殖的抑制作用,其抗病毒活性为1.88×106IU/L,是未经密码子改造菌株表达产物抗病毒活性的5.6倍。用MTT法检测PoIFN-β对猪外周血淋巴细胞成熟的影响,结果表明,猪重组PoIFN-β能有效刺激淋巴细胞转化,具有良好的免疫学活性。In order to improve expression of porcine interferon beta（PoINF-β） in Pichia pastori,the gene encoding PoIFN-β was modified for adapting codons of Pichia pastori and the terminator codon was changed from TGA to TAA.The modified gene was ligated with the secreted expression vector pPICZαC and a recombinant vector pPICZαC-PIB was developed.The plasmid pPICZαC-PIB was linearized with SacⅠ and electroporated into Pichia pastoris X-33.The recombinant colonies were identified by PCR and selected in high resistance culture.A few positive clones were obtained and one expressed the PoIFN-β protein high with a protein concentration of 258.3 mg/L.It was showed that the PoIFN-β protein concentration expressed in Pichia pastori with modified genes was 3.4 times higher than that without gene modification.The expressed supernatant was identified by SDS-PAGE and Western blotting.On the gel and membrane there were two major protein bands with molecular weight about 22 000 and 24 000 and they showed positive reaction with anti-PoIFN-β antibody.The amino acids sequence of the PoIFN-β protein was analysed by random peptide and the result demonstrated that two random peptides were completely equal with sequences among the deduced amino acids of PoIFN-β gene.The antiviral effect of PoIFN-β against vesicular stomatitis virus（VSV） was studied by the Reed-Muench method.The result indicated the antiviral activity of PoIFN-β expressed in Pichia pastori with modified genes was 1.88×106IU/L,which was 5.6 times higher than non-gene modification.The effect of PoIFN-β on the porcine lymphocyte transformation was detected by the MTT method.The result suggested that PoIFN-β could effectively stimulated the transformation of porcine lymphocyte.

关 键 词：猪β干扰素 基因修饰 毕赤酵母 抗病毒活性 MTT 

分 类 号：S852.2[农业科学—基础兽医学]