[发明专利]整合O型口蹄疫病毒shRNA转基因克隆猪的制备方法

摘要

本发明涉及一种整合O型口蹄疫病毒shRNA转基因克隆猪的制备方法，其特征在于：利用RNA干扰和转基因克隆技术制备抗O型口蹄疫病毒复制转基因克隆猪的方法，针对O型口蹄疫病毒VP1和3D基因共设计了16对shRNA,构建干扰载体16个；将干扰载体转染BHK-21细胞并对转染细胞攻毒，提取攻毒后细胞的RNA，反转录为cDNA，通过Real-timePCR筛选出两个表达质粒抑制口蹄疫病毒复制的效率最好；用组织块法分离了猪胎儿成纤维细胞，筛选出阳性细胞作为核移植的供体细胞；成功培养猪卵母细胞，利用胚胎移植将重构胚胎移入受体母猪子宫，受体母猪自然分娩获得抗口蹄疫转基因克隆仔猪，检测结果表明转基因克隆仔猪具有抑制O型口蹄疫病毒复制的能力。

主权项

一种整合O型口蹄疫病毒，其特征在于：16对抗猪O型口蹄疫病毒病shRNA的序列如下；PXL‑EGFP‑NEO‑FMDV‑VP1‑0正义链：GATCCAATTACTCAGACACTTGCAGTTTCAAGAGAACTGCAAGTGTCTGAGTAATTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAATTACTCAGACACTTGCAGTTCTCTTGAAACTGCAAGTGTCTGAGTAATTGPXL‑EGFP‑NEO‑FMDV‑VP1‑1正义链：GATCCAAGTTAATGTGTTGGACCTGATTCAAGAGATCAGGTCCAACACATTAACTTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAAGTTAATGTGTTGGACCTGATCTCTTGAATCAGGTCCAACACATTAACTTGPXL‑EGFP‑NEO‑FMDV‑VP1‑2正义链：GATCCAATGTGTTGGACCTGATGCAGTTCAAGAGACTGCATCAGGTCCAACACATTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAATGTGTTGGACCTGATGCAGTCTCTTGAACTGCATCAGGTCCAACACATTGPXL‑EGFP‑NEO‑FMDV‑VP1‑3正义链：GATCCAACAGCTTACCACAAGGAACCTTCAAGAGAGGTTCCTTGTGGTAAGCTGTTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAACAGCTTACCACAAGGAACCTCTCTTGAAGGTTCCTTGTGGTAAGCTGTTGPXL‑EGFP‑NEO‑FMDV‑VP1‑4正义链：GATCCAAGGCAGAAAGAACTCTGCCTTTCAAGAGAAGGCAGAGTTCTTTCTGCCTTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAAGGCAGAAAGAACTCTGCCTTCTCTTGAAAGGCAGAGTTCTTTCTGCCTTGPXL‑EGFP‑NEO‑FMDV‑VP1‑5正义链：GATCCAACTCTGCCTACCTCCTTCAATTCAAGAGATTGAAGGAGGTAGGCAGAGTTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAACTCTGCCTACCTCCTTCAATCTCTTGAATTGAAGGAGGTAGGCAGAGTTGPXL‑EGFP‑NEO‑FMDV‑VP1‑6正义链：GATCCAAGGCAACTCGTGTTACTGAATTCAAGAGATTCAGTAACACGAGTTGCCTTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAAGGCAACTCGTGTTACTGAATCTCTTGAATTCAGTAACACGAGTTGCCTTGPXL‑EGFP‑NEO‑FMDV‑VP1‑7正义链：GATCCAAGAGAGCCGAGACATACTGTTTCAAGAGAACAGTATGTCTCGGCTCTCTTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAAGAGAGCCGAGACATACTGTTCTCTTGAAACAGTATGTCTCGGCTCTCTTGPXL‑EGFP‑NEO‑FMDV‑3D ‑0正义链：GATCCATAGCGTCACCGAAGTTACTATTCAAGAGATAGTAACTTCGGTGACGCTATTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAATAGCGTCACCGAAGTTACTATCTCTTGAATAGTAACTTCGGTGACGCTATGPXL‑EGFP‑NEO‑FMDV‑3D ‑1正义链：GATCCAAGAGGACAAAGCGCTGTTTCTTCAAGAGAGAAACAGCGCTTTGTCCTCTTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAAGAGGACAAAGCGCTGTTTCTCTCTTGAAGAAACAGCGCTTTGTCCTCTTGPXL‑EGFP‑NEO‑FMDV‑3D ‑2正义链：GATCCAACATCATGTTCGAGGAAGTGTTCAAGAGACACTTCCTCGAACATGATGTTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAACATCATGTTCGAGGAAGTGTCTCTTGAACACTTCCTCGAACATGATGTTGPXL‑EGFP‑NEO‑FMDV‑3D ‑3正义链：GATCCAAGACTCTAGTGAACACGGAGTTCAAGAGACTCCGTGTTCACTAGAGTCTTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAAGACTCTAGTGAACACGGAGTCTCTTGAACTCCGTGTTCACTAGAGTCTTGPXL‑EGFP‑NEO‑FMDV‑3D ‑4正义链：GATCCAAGCGCATTGTTGTTGAAGGTTTCAAGAGAACCTTCAACAACAATGCGCTTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAAGCGCATTGTTGTTGAAGGTTCTCTTGAAACCTTCAACAACAATGCGCTTGPXL‑EGFP‑NEO‑FMDV‑3D ‑5正义链：GATCCAAGTGACTACGACCTGGACTTTTCAAGAGAAAGTCCAGGTCGTAGTCACTTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAAGTGACTACGACCTGGACTTTCTCTTGAAAAGTCCAGGTCGTAGTCACTTGPXL‑EGFP‑NEO‑FMDV‑3D ‑6正义链：GATCCAAGGCCTCTTCGAGATTCCAATTCAAGAGATTGGAATCTCGAAGAGGCCTTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAAGGCCTCTTCGAGATTCCAATCTCTTGAATTGGAATCTCGAAGAGGCCTTGPXL‑EGFP‑NEO‑FMDV‑3D ‑7正义链：GATCCAAGCTACAGATCACTTTACCTTTCAAGAGAAGGTAAAGTGATCTGTAGCTTTTTTTTACGCGTG反义链：AATTCACGCGTAAAAAAAAGCTACAGATCACTTTACCTTCTCTTGAAAGGTAAAGTGATCTGTAGCTTG。