摘要
根据已发表的编码牛乳铁蛋白肽（LfcinB）25个氨基酸的mRNA序列，设计了4条用于合成牛乳铁蛋白肽全基因的引物，用重叠延伸PCR法合成149bp的牛乳铁蛋白肽基因（包括牛乳铁蛋白信号肽序列、上下游酶切位点和终止密码子），并将此基因克隆到载体pI-B，获得了乳腺特异性表达质粒pI-BL，该质粒经生理盐水稀释后直接注入稳定泌乳期奶山羊和奶牛乳腺组织（注射量为400μg），以琼脂板溶圈法检测奶样抑菌活性。结果显示，注射后3～48h，奶样有明显抑菌活性，其中3～9h抑菌活性最高。
The gene encoding Lactoferriein B was artificially synthesized by overlap extension PCR. Proper restriction enzyme sites were added to it.In order to express laetoferriein B in animal mammary glands, marmnary-tissue-specifie expression vector containing 5＇flanking regulation sequence of goat β-casein and bovine growth hormone polyadenylation signal was constructed based on eukaryotie expression vector pIRESl-neo. Recombinant vectors were injected directly into the lactating mammary glands of goats and bovines. The relative milk samples were checked by Trieine-SDS-PAGE, RP-HPLC and Kirby-Bauer. The results of these tests showed that the marmnary-tissue-specifie expression vector driven by the goat beta-casein gene promoter could efficiently direct the expression of lactoferricin B in animal milk. The product exhibited evident bactericidal activity to Staphylococcus aureus and Escherichia coli.
出处
《天然产物研究与开发》
CAS
CSCD
2006年第2期229-233,共5页
Natural Product Research and Development
基金
国家自然科学基金项目(30471280)
关键词
乳铁蛋白
乳铁蛋白肽
基因合成
乳腺特异性表达
lactoferrin
lactoferricin
gene synthesis
mammary tissue specific expression
