Study On Transgenic Mice Of Mammary Gland Specific Expressing Recombinant Human Lysozyme

Lysozyme, which is also named muramidase, is an alkaline enzyme which couldhydrolysed mucopolysaccharides of pathogenic bacterium. Lysozyme is an antibacterialfactor for innate immunity. It can combine negatively charged viral protein,and also canform complex compound with DNA, RNA or apoprotein.So,lysozyme hasbactericidal,anti-inflammatory,anti-virus effect. It exists in many tissues and mucus forhuman and animal, as well as plant and microorganism. In human milk, lysozyme is oneof the most important antibacterial factors, and its content is about0.5mg/mL, which is1500-4000times in human higher than in other mammal eg: bovine，sheep and goat, andits antibacterial activity is300times higher than that of bovine. Most gram-positive and afew gram-negative bacteria are killed by lysozyme from milk, which is helpful for infantsto increase in beneficial microorganism and in resistance on disease. With the rapidsocial development,when increase in milk consumptions, the quality of milk is more andmore concerned. Genetically modify some mammal by biological technology, milkproduced through mammary gland bioreactor may become similar to human milk, whichhas nutritional and medical function. Resarching on transgenic mice mammary glandspecific expressing recombinant human lysozyme can establish the working foundationfor future milk humanized and the application of mammary gland bioreactor.This research designed and constructed two kinds of eukaryotic expression vectorspMarBLGprobianhlyts and pMarBLGpro2bianhlyts. The pMarBLGprobianhlyts vectorcontained matrix attachment region (Mar) sequence, Part of the beta lactoglobulinpromote（rupstream5kb of the transcription start site），all transcription uint sequence and3’flanking sequence of Beta lactoglobulin gene. Its MCS just located at the5’ side of thetranslation start site of BLG gene; The structrure of pMarBLGpro2biants is same topmarBLGprobianhlyts,except that the5’end of the BLG gene promoter was deleted1kbsequence, which may content negative regulation element and may suppressheterologous expression.After microinjection, for the pMarBLGprobianhlyts vector we obtained4transgenepositive founder:#1（♂）,#2（♀）,#11（♂）and#42（♂）by PCR identification,Genetically transgenic positive rate is about6.7%. F1mice for every founder line weredetected by PCR and the positive transgenic mice were gotten. Western blotting analysissuggested that human lysozyme could be detected in the positive transgenic founder and F1positive maternal mouse milk.Enzyme activity assay show recombinant lysozyme arebiological active.After microinjection, for the pMarBLGpro2bianhlyts vector we obtained#5（♂）,#9（♀）,#10（♂）and#12（♂）of four founder positive transgenic mouse by PCRidentification, Genetically transgenic positive rate is about5.0%. F1mice for everyfounder line were also detected by PCR and the positive transgenic mice were gotten.Western blotting analysis suggested that human lysozyme expression could be detectedin the positive transgenic founder and F1positive maternal mouse milk. Meanwhile,enzyme activity assay show recombinant lysozyme are biological active.