人溶菌酶cDNA的克隆及其在小鼠体内的表达

根据已发表的人溶菌酶 (human lysozyme,h L YZ) m RNA序列设计引物 ,以人乳腺第 1链 c DNA为模板 ,用 PCR扩增出 1.5 kb h L YZ双链 c DNA,其推导的氨基酸序列与国外发表的从胎盘、巨噬细胞和结肠中克隆的 h L YZ氨基酸序列同源性为 10 0 % ,与从人组织细胞中克隆的 h L YZ仅有 1个氨基酸差异 ,但与从中国人胎盘中克隆的 h L YZ具有6个氨基酸差异。将此 c DNA克隆入乳腺特异表达载体 ,用所获得的基因构件注射哺乳期小鼠 ,经 RT- PCR和微球菌溶解试验证明 ,上述基因构件能有效地驱动目的基因在小鼠乳腺中表达 ,表达量为 6 9.3mg/ L,表达具有较好的组织特异性。这些试验结果表明 ,本研究构建的表达 h L YZ c DNA的基因构件可以用于乳腺生物反应器的研制。

Cloning of Human Lysozyme cDNA and Its in vivo Expression in Mouse

To generate transgenic animal bioreactors expressing human lysozyme(hLYZ),a hLYZ cDNA of 1.5 kb was amplified by RT-PCR from the pooled first-stranded cDNA of human mammary gland using a pair of primers based on the previously published cDNA sequence for hLYZ.The cDNA was subcloned into pGEM-T vector and submitted to sequence analysis.Amino acid sequence alignment showed that the cDNA was 100% identical to that cloned from human placenta,macrophages and colon,and differed by 1 amino acid and 6 amino acids,respectively,from that cloned from histocytes and Chinese placenta.The cDNA was subcloned into a mammary gland-specific expression vector and the resulting recombinant vector pCXLYZ was injected to milking mice via tail vein route.RT-PCR showed that the hLYZ cDNA was transcribed in the mammary gland and spleen,but not in heart,liver,and kidney of the injected mice.Micrococcal lysis assay of milk from the injected mice demonstrated that the cDNA was efficiently expressed in the mammary gland with an expression level of 69.3 mg/L.These data indicate that the construct may be used for generation of animal mammary gland bioreactors expressing hLYZ.