摘要
目的 研究自行构建的动物乳腺特异表达载体p2 0 5C3的表达特性。方法 将人溶菌酶 (hLYZ)cDNA插入p2 0 5C3载体 ,用获得的基因构件注射小鼠受精卵 ,用PCR和Southernblot对出生鼠进行基因整合检测 ,用微球菌溶解试验和Westernblot对表达产物进行鉴定。结果 共出生 136只F0 代小鼠 ,从中筛选出 4只 (1♀ 3♂ )转基因整合阳性鼠 ,其中的 1只母鼠不表达hLYZ ,1只雄鼠的 4只F1 代母鼠乳汁中hLYZ的表达量分别为 5、75、175和 2 0 0 μg ml,纯合后的 3只F3代母鼠乳汁中hLYZ的表达量分别为 5 2 6 μg ml、6 4 8μg ml和 75 0 μg ml,表达仅限于乳腺中 ,表达产物的相对分子质量与正常hLYZ相同。结论 p2 0 5C3能驱动hLYZcDNA在小鼠乳腺中特异和高效表达 ,可以用于动物乳腺生物反应器的研制。
Objective To study the expression properties of self-constructed mammary gland-specific expression vector p205C3. Methods Human lysozyme (hLYZ) cDNA was subcloned into p205C3 and the recombinant vector was used to generate transgenic mice. Transgene integration was detected by PCR and confirmed by Southern blotting. Tissue specificity of the transgene expression was shown by RT-PCR and the expressed product was identified by micrococcal lysis assay and Western blotting. Results A total of 136 F 0 mice were obtained, of which 4(1♀3♂) carried the transgene. Among them, the only one female founder did not express the gene of interest and four F 1 offspring of one male founder expressed hLYZ activities in their milk with the level of 5-200 μg/ml. The expression level of F 3 homozygotes was over 500μg/ml. The expression was restricted to mammary gland and the expressed protein had the same molecular weight to that of normal hLYZ. Conclusion The self-constructed vector can efficiently drive hLYZ cDNA expression in murine mammary gland and thus useful for generation of animal mammary gland bioreactors.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2004年第2期142-145,共4页
Chinese Journal of Microbiology and Immunology
基金
国家教育部骨干教师培养计划 ( 2 0 0 0 )
江苏省高新技术 (BJ2 0 0 13 15 )资助项目
