摘要
目的:建立暂态表达人溶菌酶(hLYZ)的鸡输卵管生物反应器,以寻求一种生产药用hLYZ的有效方法。方法:将hLYZcDNA克隆入鸡输卵管特异表达载体pOV1和pOV2,将获得的重组表达载体pOV1LYZ和pOV2LYZ与一定比例的聚乙烯亚胺混合,经翅静脉注射产蛋鸡,用微球菌平板溶菌试验检测蛋清中的溶菌酶活性。结果:2个重组载体注射鸡的蛋清中均有rhLYZ的表达,pOV2LYZ的表达水平及维持时间优于pOV1LYZ。设立4个试验组,分别以不同剂量或注射次数的pOV2LYZ注射产蛋鸡,蛋清中重组酶的定量测定结果表明,1mg/2次注射组的表达效果较好,最高表达量达750μg/mL蛋清,有效表达维持7d左右,载体注射对鸡的产蛋等生理指标无明显影响。当初次载体注射鸡蛋清中的rhLYZ含量下降到注射前水平时,用同样的方法和剂量进行再次注射。结论:蛋清中的rhLYZ表达水平较初次载体注射有所提高,表达维持时间有所延长。用hLYZ特异抗体进行的Western印迹结果显示,表达在蛋清中的rhLYZ相对分子质量与天然hLYZ相同。
Objective： To test the feasibility of expressing human lysozyme（hLYZ） in hen oviduct. Methods： hLYZ cDNA was subcloned into oviduct-specific expression vectors pOV1 and pOV2, and the resultant recombinant vectors pOV1LYZ and pOV2LYZ were injected into laying hens via wing vein route. The recombinant protein was detected qualitatively and quantitatively. To investigate the dose- and/or injection time-dependent expression of pOV2LYZ vector in oviduct cells, 16 laying hens were divided into 4 groups and each was injected with 2, 3 or 4 mg of pOV2LYZ mixed with PEI. Results： Lysoplate assay and Western blotting of egg white of vector-injected hens showed that hLYZ cDNA was correctly expressed in oviduct epithelial cells and the rhLYZ was secreted efficiently into egg white. Quantitative detection of rhLYZ expression by RBB-R-labeled colorimetric assay showed that expression level of pOV2LYZ was higher than that of pOV1LYZ and thus was selected for further expression studies. Following once or twice injection of each hen with 2 mg of pOV2LYZ, secretion of hLYZ into egg white reached a maximum level of 750 μg/mL at day 5 post-injection and de- tectable expression of hLYZ lasted for 10 days. The expression level was not increased by injection with 3 or 4 mg of the pOV2LYZ vector, but overt side effect such as lowed egg production was observed. When the expression of rhLYZ dropped to its prior-injection level, the hens were re-injected with same dose of pOV2LYZ, and even higher and longer expression of rhLYZ was obtained in egg white of the vector-injected hens. Conclusion： These results indicate that chicken oviduct-specific expression vector can be used to express usable amounts of rhLYZ.
出处
《生物技术通讯》
CAS
2006年第4期546-549,共4页
Letters in Biotechnology
基金
江苏省高新技术发展计划项目(BJ2001315)
