本文選題：rhPA + 體細(xì)胞核移植�。� 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：目前,臨床上使用的溶栓藥有尿激酶、人組織纖溶酶原激活劑等,纖溶酶原激活劑類溶栓藥已經(jīng)發(fā)展到第3代,以重組人纖溶酶原激活劑為代表。重組人纖溶酶原激活劑(rhPA)是天然t-PA重組突變體,保留了 K2和P區(qū),提高了溶栓效果、纖維蛋白的親和力特異性高、半衰期延遲、使用劑量減少、全身出血和顱內(nèi)出血發(fā)生率降低等特點(diǎn)。利用乳腺生物反應(yīng)器生產(chǎn)rhPA,大大提高生產(chǎn)量、目的蛋白質(zhì)可以在機(jī)體內(nèi)翻譯,磷酸化、羧化等修飾,生物活性高,成本低,樣品純化簡(jiǎn)單等優(yōu)點(diǎn)。通過沉淀法、超濾法、層析法等技術(shù)相結(jié)合,有效的從乳汁中分離和純化到表達(dá)蛋白。本實(shí)驗(yàn)通過體細(xì)胞核移植技術(shù)制備了乳腺特異性表達(dá)rhPA轉(zhuǎn)基因山羊,重組人纖溶酶原激活劑cDNA在山羊乳腺中特異性表達(dá)。通過ELISA、Western Blot,檢測(cè)rhPA的表達(dá)產(chǎn)物分泌到乳汁內(nèi);經(jīng)FAPA檢測(cè),表達(dá)產(chǎn)物有較高的比活性。rhPA轉(zhuǎn)基因山羊制備:無(wú)菌手術(shù)取30日齡的羊胎兒,分離提取山羊胎兒成纖維細(xì)胞培養(yǎng),電轉(zhuǎn)染乳腺特異性表達(dá)載體BCL14/rhPA基因到胎兒成纖維細(xì)胞。用含有G418600μg/mL的細(xì)胞培養(yǎng)液篩選7-10天后,挑取單克隆細(xì)胞株經(jīng)PCR檢測(cè)篩選出陽(yáng)性細(xì)胞株24株,其中選取7株作為供核細(xì)胞。用含0.5%FBS的DMEM/F12培養(yǎng)液饑餓72h陽(yáng)性細(xì)胞株作為供核細(xì)胞。活體輸卵管沖洗法獲取MII期的卵母細(xì)胞,去核后作為核受體進(jìn)行體細(xì)胞核移植,重構(gòu)胚移植到同期發(fā)情的受體羊輸卵管內(nèi)。30天、60天進(jìn)行B超檢查受體羊的妊娠情況,出生的小羊PCR檢測(cè)是否整合rhPA基因。超數(shù)排卵供體羊42只,獲得MII期卵母細(xì)胞438枚,受體羊26只,移植重構(gòu)胚256枚,7只受體羊妊娠,其懷孕率為26.9%,出生2只小羊,提取基因組進(jìn)行PCR檢測(cè)為rhPA轉(zhuǎn)基因山羊(BP21、BP22)。獲得的轉(zhuǎn)基因山羊進(jìn)行飼養(yǎng)、性成熟后,與正常公羊配種、妊娠、分娩,出生的小羊提取基因組,PCR檢測(cè)為rhPA轉(zhuǎn)基因山羊。收集乳汁,高速離心取乳清,ELISA免疫原性檢測(cè),半定量分析轉(zhuǎn)基因山羊乳清中rhPA表達(dá)量約是92.2μg/mL。SDS-PAGA電泳和Western Blot檢測(cè)克隆羊乳清,出現(xiàn)了約39kD大小的特異性目的條帶。FAPA與ELISA檢測(cè)結(jié)合顯示,BP21轉(zhuǎn)基因羊乳清中所含rhPA的比活性大約為阿替普酶的40倍。表達(dá)產(chǎn)物rhPA的分離和鑒定:羊乳經(jīng)過超速離心、酸堿沉淀去除脂肪和酪蛋白后,上樣到賴氨酸親和層析株進(jìn)行分離和純化。SDS-PAGA電泳和Western Blot檢測(cè)有純化樣品中有39kDa的特異性目的條帶,FAPA檢測(cè)具有溶栓活性,說明羊乳經(jīng)過L-賴氨酸親和層析純化后依然具有溶栓活性。本實(shí)驗(yàn)利用體細(xì)胞核移植技術(shù)獲得轉(zhuǎn)rhPA山羊,乳汁中rhPA特異性表達(dá)含量為大約73.6μg/mL,具有高比活性。表達(dá)產(chǎn)物能夠通過L-賴氨酸親和層析分離和純化出來(lái),純化產(chǎn)物回收率高、生物學(xué)活性高,為后續(xù)進(jìn)一步純化奠定基礎(chǔ)。目前未見關(guān)于重組人纖溶酶原激活劑在山羊乳腺上表達(dá)的相關(guān)報(bào)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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S827

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本文編號(hào)：1895380