Preparation Of Recombinant Human Lactoferrin Transgenic Dairy Goats And Extraction Of Expression Products And Antibacterial Activity

Lactoferrin(LF)as an iron binding glycoprotein has been used in medicine?food and skincare production because of its antimicrobial activity.However,the natural LF currently marketed is purified from cow milk.The output of natural LF cannot satisfy expanding demand in market,due to its limited source.Therefore,utilizing transgenic technology to produce Recombinant Human Lactoferrin(rhLF)as a substitute of natural LF has gradually become main pathway.In this study,mammary gland-specific expression plasmid vector PCL25/hLF which was saved by our own laboratory was amplified.And transgene(zpz-1)with enzyme digestion of PCL25/hLF plasmid was obtained by Sal I and Not I.Utilizing microinjection technology to inject vector zpz-1 into female goat fetal fibroblast cell(N31).At last,16 monoclonal cell lines were acquired by screening with G418.And 7 of these monoclonal cell lines integrated PCL25/hLF gene identified by PCR.Utilizing nuclear transfer technology and electro-fusion technology,and using N31zpz-1-13 monoclonal cell(1 of 7 monoclonal cell lines integrating PCL25/hLF gene)as donor cell,140 reconstruction embryos were acquired and 136 reconstruction embryos were transplanted into 9 surrogate goats.And 22 weeks after transplanting,2 of 9 surrogate goats respectively produced two female goats(numbered zp-71 and zp-72).However,only zp-71 grown to adulthood.After identifying by PCR,zp-71 integrated transgene PCL25/hLF.When transgenic goat zp-71 come into estrus,mating with normal male goat.Finally,we delivered it after 22 weeks.Milk collecting was continuous for 30 days.Using ELISA to detect rhLF expression in whey of transgenic goat zp-71.AKATA protein purification system and cation-exchange chromatography were used to extract rhLF from the milk.After that,using penicillin-streptomycin and concentrated human colostrum as positive control,normal goat milk as negative control,concentrated whey and rhLF(?lmg)extracted from transgenic goat milk as experimental samples for Bacterial Inhabition Ring Test.The results showed that the whey of zp-71 contained rhLF in continuous 30 days by ELISA.Compared with wildtype hLF,the rhLF PCL25/hLF expression vector(containing ?-casin and CMV chimeric promoters)can be expressed in mammary gland of transgenic goat effectively and stabilityly.In the progress of cation-exchange chromatography,two elution peaks were acquired by using eluent of 0.5M NaCl-HAC-Tris.And the SDS-PAGE electrophoresis showed that elution peak I contained many bands,but the extractive of elution peak ? was pure.The Western Blotting showed that the extractive of elution peak II was rhLF.The Bacterial Inhabition Ring Test showed that the whey of transgene goat zp-71 and the isolated protein rhLF all had antimicrobial activity,and the diameters of inhibition rings after cultivating 4 hours were 1.70cm and 1.30cm.This study indicated that transgene PCL25/hLF was expressed in mammary gland of transgenic dairy goat.Moreover,the rhLF can be extracted from the milk of transgenic goat.Both transgenic goat whey and extractive rhLF have antimicrobial activity.All the results confirmed that rhLF is solely exist in transgenic goat milk,and its bacteriostatic function is similar with wildtype hLF.Meanwhile,this study also first showed that HAC-Tris-NaCl buffer solution could effectively elute high-purity rhLF from cation-exchange column.And this purification method would be the first report according to literatures at home and abroad.In this study we successfully established and confirmed a complete useful scheme to isolate rhLF from transgenic goat milk.And we hope that all the methods and results in this study could be used or give a help in future industrialized purification.