【作者】 薛方明；

【导师】 孙怀昌；

【作者基本信息】 扬州大学 ， 动物学， 2005， 硕士

【摘要】 奶牛乳腺炎是最常见、防治最难、花费最多的奶牛疾病之一,至今仍无有效的根治手段。目前奶牛乳腺炎的防治主要依赖抗菌素等常规药物,虽有一定的效果,但也日益暴露出抗菌素残留和耐药菌株流行等危害人畜健康等严重问题。近年来,虽然不断有用新的药物或方法进行奶牛乳腺炎防治的尝试,但目前还没有真正能替代抗菌素的有效药物。 前期研究结果表明,含人溶菌酶(hLYZ)基因的乳腺特异表达载体不仅能在动物乳腺中表达,而且表达的重组酶对金黄色葡萄球菌、链球菌和大肠杆菌等奶牛乳腺炎常见致病菌具有明显的抑制作用,重组载体注射对奶牛乳腺炎具有明显的治疗效果,相关研究成果已获国家发明专利(专利号:ZL02148545.3)。 为了进一步提高基因药物治疗奶牛乳腺炎的效果和安全性,本研究对原重组载体进行了优化和改造,获得新的重组载体命名为乳腺特异表达载体p215C3LYZ,并用hLYZ基因构建成真核表达载体pcDNAKLYZ,将获得的重组载体用于奶牛乳腺炎的预防和干乳期的治疗。经体内外试验证明两种重组载体均能获得正确表达,采用不同注射途径、剂量、次数等方案,将两种重组载体分别注射奶牛乳腺,对其乳汁中暂态表达的重组人溶菌酶含量及动态变化进行了系统测定,建立了行之有效的重组载体注射方法,为临床治疗奶牛乳腺炎提供了依据。 本研究将乳腺特异表达和真核表达重组载体分别用于泌乳期奶牛乳腺炎的治疗和干乳期的预防,田间试验结果表明,两种载体在不同阶段对奶牛乳腺炎的防治均有较好的疗效。此外,对基因药物的安全性问题进行了初步探讨,进一步证实了该基因药物对动物、人类及环境等的安全性。同时,对基因药物的开发应用进行了初步研究,结果表明,该基因药物的生产开发符合相应的规定、标准。由此可见,该基因药物不仅在实际应用中具有安全可靠的疗效,而且在大规模生产开发方面也具有很好的可行性。因此,该基因药物可以作为替代抗菌素等常规药物用于防治奶牛乳腺炎的新型药物加以开发应用。更多还原

【Abstract】 Bovine mastitis is the most costly disease in the dairy industry and can be caused by at least 135 different agents, mostly bacteria. Antibiotic treatment has been widely used for mastitis treatment and control, but it has been a hotly debating issue all over the world concerning drug-resistant bacteria and the safety of residue antibiotics to human health.To optimize the genetic drug for treating bovine mastitis, the cDNA for human lysozyme (hLYZ) was subcloned into a mammary gland-specific expression vector p215C3 and a eukaryotic expression vector pcDNAK, respectively. The three recombinant expression vectors p205C3LYZ, p215C3LYZ and pcDNAKLYZ were injected into lactating mice via vein route. Micrococcal lysis assay and Remazol Brilliant Blue R (RBB-R)-labeled colorimetric assay of the collected milk samples showed that rhLYZ was efficiently expressed in the mammary glands.To test whether the recombinant vectors can express active lysozyme in bovine mammary glands, eukaryotic expression vector pcDNAKLYZ or p215C3LYZ was injected into milk cisterna of the normal mammary gland by different means and expression of gene of interest was demonstrated by using RBB-R-labeled colorimetric assay. The results indicate that both the two recombinant vectors can express active enzyme in milk, which lasted for at least 20 days. The treatments with different routes, doses and injection times showed that effectiveness of quarter base injection was better than that of intracisternal injection, which was dose- and injection time-dependent. Fermentation experiments showed that the low level of recombinant lysozyme expressed in milk had no significant influence on milk fermentation and thus yogurt production.To evaluate the feasibility of recombinant vecotrs for treating dairy cow mastitisresistant to antibiotic treatment, the mammary gland-specific expression vector p215C3LYZ was injected into the milk cisterna or base of each quarter of the dairy cows with clinical or subclinical disease and the effectiveness of the treatment was evaluated on the basis of clinical changes and milk somatic cell counting using the standard C.M.T assay. After daily injection of 400(μg of the recombinant vector for successive 2 days, >90% of the treated cows become clinically normal with about 30% cure rate at 10 day post-treatment.To explore the feasibility of preventing cow mastitis during drying off, another eukaryotic expression vector pcDNAKLYZ was injected into the base of each quarter of the dairy cows. The result showed that the prevention effectiveness of the genetic drug was better than that of antibiotic treatment.To evaluate the biosafety of recombinant vectors for clinical use, different doses of the recombinant vector were injected into mice, calves and dairy cows with mastitis, respectively, and its dynamics of degradation in mouse stomach was investigated by agarose gel electrophoresis, polymerase chain reaction and transformant isolation from feces samples. The results showed that injection of the vector had no obvious side effect on the animals tested. The vector was not detectable in mouse stomach one minute after oral administration even by sensitive PCR and no vector-transformant was isolated from the feces samples. These data indicate that the recombmant vector is safe for dairy cow use without evidence of horizontal transmission and thus can be further developed as a new drug for dairy cow mastitis.These results indicate that injection of the recombinant plasmid containing hLYZ cDNA can be used as an alternative strategy for treating and preventing dairy cow mastitis.更多还原