基因治疗奶牛乳腺炎的生物安全评价

为评价基因治疗奶牛乳腺炎的生物安全性,以3头患隐性乳腺炎的奶牛作为试验对象,通过乳房基部穿刺注射接种携带人溶菌酶基因的乳腺特异性表达重组质粒p215C3LYZ(1 600μg·头~(-1)),24 h后以相同剂量重复注射1次.定期采集试验奶牛的乳样、粪样、尿样以及试验牛舍的垫料和水样,并利用建立的PCR进行检测.结果发现,除第2次注射24 h内可从奶样中检测到重组质粒外,其他样品均为阴性,这表明重组质粒p215C3LYZ不能在奶牛体内长期残留,也不能在环境中释放及散播.所有奶牛均于第2次注射20 d后处死,并采集血液、心、肝,脾、肺、肾、肌肉以及乳腺等样品进行PCR检测,结果表明:重组质粒p215C3LYZ在奶牛体内未发生转移.

Biosafety evaluation of gene therapy on bovine subclinical mastitis

To evaluate the clinical biosafety of gene therapy on bovine mastitis,three lactating cows with subclinical mastiffs(determined by using the standard California mastitis test,CMT)were assigned to the tested group,and three healthy lactating cows(characterized by udder redness,swelling,hardness,pain and grossly abnormal milk)were set to be control.Each experimental animal was treated with 1 600μg(400 μg each quarter)of the mammary-specific expression vector p205C3LYZ described elsewhere,used the 5'-and 3'-regulatory elements of goat β-lactoglobulin(blg)gene and the first intron of the β-casein gene of dairy goat to control hLYZ cDNA expression and bla gene as the selection marker,and boosted in the same way with the same dose 24 hours after the priming.Milk,feces,urine,litte,water samples and the tissues of heart,liver,spleen,lung,kidney,muscle and galactophore(20 days after the boosting treatment)were collected and submitted to PCR detection.The data showed that no recombinant plasmid p215C3LYZ was detected,except for the milk samples within 24 hours following the boosting treatment.This suggested that the gene therapy on bovine mastitis by using p205C3LYZ was safe in clinic with no transformation in different tissues and no dissemination in the entironment.