重组型rhPA在山羊乳腺中特异性表达及产物分离与分析

发布时间：2018-05-16 04:10

本文选题：rhPA + 体细胞核移植　；参考：《扬州大学》2017年硕士论文

【摘要】：目前,临床上使用的溶栓药有尿激酶、人组织纤溶酶原激活剂等,纤溶酶原激活剂类溶栓药已经发展到第3代,以重组人纤溶酶原激活剂为代表。重组人纤溶酶原激活剂(rhPA)是天然t-PA重组突变体,保留了 K2和P区,提高了溶栓效果、纤维蛋白的亲和力特异性高、半衰期延迟、使用剂量减少、全身出血和颅内出血发生率降低等特点。利用乳腺生物反应器生产rhPA,大大提高生产量、目的蛋白质可以在机体内翻译,磷酸化、羧化等修饰,生物活性高,成本低,样品纯化简单等优点。通过沉淀法、超滤法、层析法等技术相结合,有效的从乳汁中分离和纯化到表达蛋白。本实验通过体细胞核移植技术制备了乳腺特异性表达rhPA转基因山羊,重组人纤溶酶原激活剂cDNA在山羊乳腺中特异性表达。通过ELISA、Western Blot,检测rhPA的表达产物分泌到乳汁内;经FAPA检测,表达产物有较高的比活性。rhPA转基因山羊制备:无菌手术取30日龄的羊胎儿,分离提取山羊胎儿成纤维细胞培养,电转染乳腺特异性表达载体BCL14/rhPA基因到胎儿成纤维细胞。用含有G418600μg/mL的细胞培养液筛选7-10天后,挑取单克隆细胞株经PCR检测筛选出阳性细胞株24株,其中选取7株作为供核细胞。用含0.5%FBS的DMEM/F12培养液饥饿72h阳性细胞株作为供核细胞。活体输卵管冲洗法获取MII期的卵母细胞,去核后作为核受体进行体细胞核移植,重构胚移植到同期发情的受体羊输卵管内。30天、60天进行B超检查受体羊的妊娠情况,出生的小羊PCR检测是否整合rhPA基因。超数排卵供体羊42只,获得MII期卵母细胞438枚,受体羊26只,移植重构胚256枚,7只受体羊妊娠,其怀孕率为26.9%,出生2只小羊,提取基因组进行PCR检测为rhPA转基因山羊(BP21、BP22)。获得的转基因山羊进行饲养、性成熟后,与正常公羊配种、妊娠、分娩,出生的小羊提取基因组,PCR检测为rhPA转基因山羊。收集乳汁,高速离心取乳清,ELISA免疫原性检测,半定量分析转基因山羊乳清中rhPA表达量约是92.2μg/mL。SDS-PAGA电泳和Western Blot检测克隆羊乳清,出现了约39kD大小的特异性目的条带。FAPA与ELISA检测结合显示,BP21转基因羊乳清中所含rhPA的比活性大约为阿替普酶的40倍。表达产物rhPA的分离和鉴定:羊乳经过超速离心、酸碱沉淀去除脂肪和酪蛋白后,上样到赖氨酸亲和层析株进行分离和纯化。SDS-PAGA电泳和Western Blot检测有纯化样品中有39kDa的特异性目的条带,FAPA检测具有溶栓活性,说明羊乳经过L-赖氨酸亲和层析纯化后依然具有溶栓活性。本实验利用体细胞核移植技术获得转rhPA山羊,乳汁中rhPA特异性表达含量为大约73.6μg/mL,具有高比活性。表达产物能够通过L-赖氨酸亲和层析分离和纯化出来,纯化产物回收率高、生物学活性高,为后续进一步纯化奠定基础。目前未见关于重组人纤溶酶原激活剂在山羊乳腺上表达的相关报道。
[Abstract]:At present, urokinase, human tissue plasminogen activator and so on are used in clinical thrombolytic agents. Plasminogen activators have been developed to the third generation, represented by recombinant human plasminogen activators. Recombinant human plasminogen activator (rhPA) is a natural mutant of t-PA, which preserves K2 and P regions, improves thrombolytic effect, and has high affinity specificity, delayed half-life and reduced dosage of fibrin. The incidence of systemic hemorrhage and intracranial hemorrhage was decreased. The production of rhPAusing mammary gland bioreactor can greatly improve the production. Objective protein can be modified in vivo, phosphorylation, carboxylation, high biological activity, low cost, simple sample purification and so on. By means of precipitation, ultrafiltration, chromatography and other techniques, the expressed protein was effectively isolated and purified from milk. In this experiment, mammary gland specific expression of rhPA transgenic goat was prepared by somatic cell nuclear transfer technique, and recombinant human plasminogen activator cDNA was expressed in goat mammary gland. The expression product of rhPA was secreted into the milk by Elisa Western blot.The FAPA assay showed that the expressed product had higher specific activity. The goat was prepared by sterility operation, and the goat fetal fibroblasts were isolated and cultured. Breast specific expression vector BCL14/rhPA gene was transfected into fetal fibroblasts. After 7-10 days of screening with G418600 渭 g/mL, 24 positive cell lines were screened by PCR, and 7 of them were selected as donor cells. DMEM/F12 culture medium containing 0.5 S was used as donor cells for 72 h after starvation. Oocytes of MII stage were obtained by living oviduct washing method. Somatic cell nuclear transfer was performed as nuclear receptor after nuclear removal. The reconstructed embryos were transplanted into oviduct of estrous recipient sheep for 60 days. The pregnancy of recipient sheep was examined by B-ultrasound. Born lambs were tested for the integration of rhPA gene by PCR. In 42 superovulation donor sheep, 438 oocytes and 26 recipient sheep were obtained in MII phase, and 256 reconstructed embryos were transplanted into 7 recipient sheep. The pregnancy rate was 26.9 and 2 lambs were born. The genomic PCR was extracted and detected as rhPA transgenic goat BP21 and BP22. The transgenic goats were bred. After sexual maturation, they were bred with normal male sheep, pregnant, parturition, and newborn lambs were extracted and detected to be rhPA transgenic goats by genomic DNA polymerase chain reaction (PCR). The milk was collected, the immunogenicity of whey was detected by Elisa, and the expression of rhPA in the whey of transgenic goat was about 92.2 渭 g/mL.SDS-PAGA electrophoretic and Western Blot was used to detect the whey of cloned sheep. A specific target band of about the size of 39kD. FAPA combined with ELISA detection showed that the specific activity of rhPA in the milk of BP21 transgenic sheep was about 40 times as high as that of atropase. Isolation and Identification of expression Product rhPA: goat milk after ultracentrifugation, acid-base precipitation to remove fat and casein, SDS-PAGA electrophoresis and Western Blot were used to detect 39kDa in purified samples. The results showed that goat milk still had thrombolytic activity after purification by L-lysine affinity chromatography. In this experiment, rhPA goats were obtained by somatic cell nuclear transfer technique. The specific expression of rhPA in milk was about 73.6 渭 g / mL, which showed high specific activity. The expressed product can be separated and purified by L- lysine affinity chromatography. The purified product has high recovery rate and high biological activity, which lays a foundation for further purification. There is no report on the expression of recombinant human plasminogen activator in goat mammary gland.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S827

【参考文献】