人溶菌酶基因密码子优化及其在小鼠乳腺中的表达

Expression of a codon-optimized human lysozyme gene in mouse milk

人溶菌酶是人体内的一种非特异性免疫物质,具有杀菌消炎、免疫调节、改善胃肠道菌群等作用,在抗生素替代方面具有广阔的应用前景。已报道人溶菌酶基因c DNA在内源的αS2酪蛋白基因启动子调控下的表达量不高(23~31μg/m L),特尝试密码子优化策略以改善表达量。优化了人溶菌酶基因c DNA的密码子,连入以小鼠乳清酸蛋白(m WAP)基因座序列为调控元件的乳腺表达载体,验证其表达效率。通过原核注射制备转基因小鼠,PCR检测发现,出生的21只小鼠中有4只为转基因阳性,且能按孟德尔遗传规律传递给下一代;Western blot检测表明,小鼠乳汁中有微量的重组人溶菌酶表达。因此,密码子优化后的人溶菌酶基因可以在小鼠乳腺中获得有效表达,这可为外源基因定点整合的人溶菌酶转基因动物的高效表达研究奠定基础。

Human lysozyme(hLZ),as a natural non-specific immune protein,plays an important role in killing bacteria,modulating the inflammatory response,influencing the composition of intestinal microbiota populations,which has broad application prospect in antibiotic replacement.It is reported that the expression of recombinant human lysozyme(rhLZ)is low(23-31 pg/mL)with the cDNA under the control of endogenous a S2 casein gene promoter,so we try the method of codon optimization to improve the expression.The cDNA sequence of hLZ was codon-optimized and inserted into pMWAP-Zeo that contains 8.5 kb 5’and 8.0 kb 3’sequence of mouse whey acid protein(mWAP)gene locus,the final expression vector was pMWAP-hLZ.The pMWAP-hLZ was injected into pronuclei of mouse fertilized embryos and 21 transgenic mice were generated.A total of 4 positive transgenic mice were identified by PCR,and transgene could all be transmitted from founder transgenic mice to their offspring according to Mendelian inheritance.Western blot analysis showed that rhLZ was expressed at low-level in the mammary gland of the transgenic mice.In conclusion,the codon optimization hLZ could get low-level expression in mouse milk.Our results suggest a potential approach to produce human lysozyme genetically modified animals with the site-specific integration of exogenous genes and high expression of recombinant protein in milk of livestock.