本發(fā)明涉及生物,具體涉及一種基于c/d?box莖環(huán)結(jié)構(gòu)的高效vlp載體及其遞送mrna的方法。
背景技術(shù):
1、vlp(virus?like?particle)技術(shù)來源于慢病毒系統(tǒng),可利用病毒的高親嗜性特性實(shí)現(xiàn)有效的細(xì)胞內(nèi)遞送,遞送對象可為核酸、蛋白質(zhì)或者核糖核蛋白(rnp)等。但是病毒在遞送核酸時,多數(shù)以dna形式遞送,且慢病毒所遞送的dna存在基因組隨機(jī)插入的情況,因此在遞送crispr/cas9系統(tǒng)時,cas9蛋白基因在體內(nèi)存在時間較久,持續(xù)翻譯成cas9蛋白,會導(dǎo)致較大機(jī)率的“脫靶”效應(yīng)(與原設(shè)計的基因剪切效應(yīng)無關(guān)的基因剪切)。在實(shí)際臨床應(yīng)用中,可將cas9以mrna或蛋白的形式瞬時遞送到人體中,以達(dá)到治療目的同時大幅降低脫靶效應(yīng)。
2、現(xiàn)有技術(shù)中公開了將ms2噬菌體衣殼蛋白(ms2c)和慢病毒gag-pol蛋白融合后表達(dá)于vlp的內(nèi)核中,利用莖環(huán)結(jié)構(gòu)(stem?loop)可與ms2c蛋白特異性識別和結(jié)合的原理,將cas9蛋白mrna和stem?loop融合后,通過stem?loop與vlp內(nèi)核中的ms2c特異性結(jié)合,將cas9蛋白mrna導(dǎo)入vlp內(nèi)核;同時將針對特定基因的grna克隆至慢病毒穿梭質(zhì)粒,最終組裝出攜帶有cas9?mrna和grna的類似慢病毒結(jié)構(gòu)的vlp。vlp將cas9?mrna和grna遞送至靶細(xì)胞的胞質(zhì)中,通過翻譯cas9蛋白,在靶細(xì)胞內(nèi)發(fā)揮出crispr-cas9系統(tǒng)的基因編輯效果。但現(xiàn)有慢病毒載體遞送rna技術(shù)中的ms2c-stem?loop的組合特異性結(jié)合效率低,導(dǎo)致cas9?mrna遞送效率偏低。
3、l7ae蛋白是一種高度保守的蛋白質(zhì),屬于l7ae蛋白家族,它能夠結(jié)合核糖體中的特殊莖環(huán)結(jié)構(gòu)c/d?box。本發(fā)明使用的vlp遞送技術(shù)是以慢病毒為載體骨架。將l7ae蛋白和慢病毒gagpol蛋白融合后表達(dá)于vlp的內(nèi)核中。利用莖環(huán)結(jié)構(gòu)c/d?box可與l7ae蛋白特異性識別和結(jié)合的原理,將cas9蛋白mrna和莖環(huán)結(jié)構(gòu)c/d?box融合后,通過c/d?box與vlp內(nèi)核中的l7ae特異性結(jié)合,將cas9蛋白mrna導(dǎo)入vlp內(nèi)核;同時將針對特定基因的grna克隆至慢病毒穿梭質(zhì)粒,最終組裝出攜帶有cas9?mrna和grna的類似慢病毒結(jié)構(gòu)的vlp。vlp將cas9mrna和grna遞送至靶細(xì)胞的胞質(zhì)中,通過短時間翻譯cas9蛋白,在靶細(xì)胞內(nèi)發(fā)揮出crispr-cas9系統(tǒng)的“瞬時”基因編輯效果,提高了vlp載體的mrna遞送效率和crispr-cas9編輯效率。
技術(shù)實(shí)現(xiàn)思路
1、本發(fā)明的目的是提供一種基于c/d?box莖環(huán)結(jié)構(gòu)的高效vlp載體及其遞送mrna的方法。
2、為實(shí)現(xiàn)上述發(fā)明目的,本發(fā)明的技術(shù)方案如下:
3、一方面,本發(fā)明提供了一種基于c/d?box莖環(huán)結(jié)構(gòu)的vlp載體,所述的vlp載體是通過將含有cas9-6×c/d?box序列的質(zhì)粒、含有l(wèi)7ae序列的質(zhì)粒轉(zhuǎn)染入目的細(xì)胞制備而得,所述的cas9-6×c/d?box序列如seq?id?no:1所示。
4、seq?id?no:1:
5、gacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggcgactaaaccggtcttgcagcacatctatgaagaaagccgctactctgaatttatgatgctgcaggtcgactctagaaactatgaagaaagccgctactctgaatttatgatgctgcagtattcccgggttcattagatcctaaggtacctaattgcctagaaactatgaagaaagccgctactctgaatttatgatgctgcaggtcgactctagaaactatgaagaaagccgctactctgaatttatgatgctgcagtattcccgggttcattagatcctaaggtacctaattgcctagaaactatgaagaaagccgctactctgaatttatgatgctgcaggtcgactctagaaactatgaagaaagccgctactctgaatttatgatg
6、具體地,所述的含有l(wèi)7ae序列的質(zhì)粒的制備包括:將pms2m-ph-gag-pol-d64v質(zhì)粒中ms2c基因替換為l7ae基因,l7ae-ph-gag-pol-d64v質(zhì)粒上l7ae-gag-pol基因序列如seqid?no:3所示。
7、seq?id?no:3:
8、ggaggcggaggcggaggcggaggcatgtacgtgagatttgaggttcctgaggacatgcagaacgaagctctgagtctgctggagaaggttagggagagcggtaaggtaaagaaaggtaccaacgagacgacaaaggctgtggagaggggactggcaaagctcgtttacatcgcagaggatgttgacccgcctgagatcgttgctcatctgcccctcctctgcgaggagaagaatgtgccgtacatttacgttaaaagcaagaacgaccttggaagggctgtgggcattgaggtgccatgcgcttcggcagcgataatcaacgagggagagctgagaaaggagcttggaagccttgtggagaagattaaaggccttcagaagagccagaactatccgattgtgcagtccggactcagatctcgagctcaagcttcgaattctgcagtcgacggtaccgcgggcccgggatccatggactcgggccgggacttcctgaccctgcacggcctacaggatgatgaggatctacaggcgctgctgaagggcagccagctcctgaaggtgaagtccagctcatggaggagagagcgcttctacaagttgcaggaggactgcaagaccatctggcaggagtcccgcaaggtcatgcggaccccggagtcccagctgttctccatcgaggacattcaggaggtgcgaatggggcaccgcacggagggtctggagaagttcgcccgtgatgtgcccgaggaccgctgcttctccattgtcttcaaggaccagcgcaatacactagacctcatcgccccatcgccagctgatgcccagcactgggtgctggggctgcacaagatcatccaccactcaggctccatggaccagcgtcagaagctacagcactggattcactcctgcttgcgaaaagctgacaaaaacaaggacaacaagatgagcttcaaggagctgcagaacttcctgaaggagctcaacatccagcctcgagccgaattcaccctggccgccagggccagcgtgctgagcggcggcgagctggacaggtgggagaagatcaggctgaggcccggcggcaagaagaagtataagctgaagcacatcgtgtgggccagcagggagctggagaggttcgccgtgaaccccggcctgctggagaccagcgagggctgcaggcagatcctgggccagctgcagcccagcctgcagaccggcagcgaggagctgaggagcctgtacaacaccgtggccaccctgtactgcgtgcaccagaggatcgagatcaaggacaccaaggaggccctggacaagatcgaggaggagcagaacaagtccaagaagaaggcccagcaggccgccgccgacaccggccacagcagccaggtgagccagaactaccccatcgtgcagaacatccagggccagatggtgcaccaggccatcagccccaggaccctgaacgcctgggtgaaggtggtggaggagaaggccttcagccccgaggtgatccccatgttcagcgccctgagcgagggagccaccccccaggacctgaacaccatgctgaacaccgtgggcggccaccaggccgccatgcagatgctgaaggagaccatcaacgaggaggccgccgagtgggacagggtgcaccccgtgcacgccggccccatcgcccccggccagatgagggagccccgcggcagcgacatcgccggcaccaccagcaccctgcaggagcagatcggctggatgaccaacaacccccccatccccgtgggcgaaatctacaagaggtggatcatcctgggcctgaacaagatcgtgaggatgtacagccccaccagcatcctggatatcaggcagggccccaaagagcccttcagggactacgtggacaggttctacaagaccctgcgcgccgagcaggccagccaggaggtgaagaactggatgaccgagaccctgctggtgcagaacgccaaccccgactgcaagaccatcctgaaggccctgggacccgccgccaccctggaggagatgatgaccgcctgccagggcgtgggcggccccggccacaaggccagggtgctggccgaggccatgagccaggtgaccaacaccgccaccatcatgatgcagaggggcaacttcaggaaccagaggaagatggtgaagtgcttcaactgcggcaaggagggccacaccgccaggaactgccgcgcccccaggaagaagggctgctggaagtgcggcaaggagggccaccagatgaaggactgcaccgagaggcaggctaattttttagggaagatctggccttcctacaagggaaggccagggaattttcttcagagcagaccagagccaacagccccaccatttcttcagagcagaccagagccaacagccccaccagaagagagcttcaggtctggggtagagacaacaactccccctcagaagcaggagccgatagacaaggaactgtatcctttaacttccctcagatcactctttggcaacgacccctcgtcacaataaagatcggtggccagctgaaggaggccctgctggacaccggcgccgacgacaccgtgctggaggagatgagcctgcccggcaggtggaagcccaagatgatcggcggcatcggcggcttcatcaaggtgaggcagtacgaccagatcctgatcgagatctgcggccacaaggccatcggcaccgtgctggtgggacctacacctgtgaacatcatcggcaggaacctgctgacccagatcggctgcaccctgaacttccccatcagccccatcgagaccgtgcccgtgaagctgaagcccggcatggacggccctaaggtgaagcagtggcccctgaccgaggagaagatcaaggccctggtggagatctgcaccgagatggagaaggagggcaagatcagcaagatcggccccgagaacccctacaacacccccgtgttcgccatcaagaagaaggacagcaccaagtggaggaagctggtggacttcagggagctgaacaagaggacccaggacttctgggaggtgcagctgggcatcccccaccccgccggcctgaagaagaagaagagcgtgaccgtgctggacgtgggcgacgcctacttcagcgtgcccctggacgaggacttcaggaagtataccgccttcaccatccccagcatcaacaacgagacccccggcatccgctaccagtacaacgtgctgccccagggctggaagggcagccccgccatcttccagagcagcatgacaaagatcctggagcccttcaagaagcagaaccccgacatcgtgatctatcagtacatggacgacctgtacgtgggcagcgacctggagatcggccagcacaggaccaagatcgaggagctgaggcagcacctgctgaggtggggcctgaccacccccgacaagaagcaccagaaggagcccccattcctgtggatgggctacgagctgcaccccgacaagtggaccgtgcagcccatcgtgctgcccgagaaggacagctggaccgtgaacgacattcagaagctggtgggcaagctgaactgggccagccagatctaccctggcatcaaggtgaggcagctgtgcaagctgctgaggggcacaaaggctctgaccgaggtgatccccctgaccgaggaggccgagctggagctggccgagaacagggagatcctgaaggagcccgtgcacggcgtgtactacgaccccagcaaggacctgatcgccgagatccagaagcagggccagggccagtggacctaccagatctaccaggagcccttcaagaacctgaagaccggcaagtacgcccgcatgcgcggcgcccacaccaacgacgtgaagcagctgaccgaggccgtgcagaagatcaccaccgagagcatcgtgatctggggcaagactcctaagttcaagctgcccatccagaaggagacctgggagacctggtggaccgagtactggcaggccacctggattcccgagtgggagttcgtgaacacccctcccctggtgaagctgtggtatcagctggagaaggagcccatcgtgggcgccgagaccttctacgtggacggcgccgccaacagggagaccaagctgggcaaggccggctacgtgaccaacaagggccgccagaaggtggtgcccctgaccaacaccaccaaccagaagaccgagctgcaggctatctacctggccctgcaggactcaggcctggaggtgaacatcgtgaccgacagccagtacgccctgggcatcatccaggcccagcccgacaagagcgagagcgagctggtgaaccagatcatcgagcagctgatcaagaaggagaaggtgtacctggcctgggtgcccgcccacaagggcatcggcggcaacgagcaggtggacaagctggtgagcgccggcatcaggaagatcctgttcctggacggcatcgacaaggcccaggacgagcacgagaagtaccacagcaactggagggctatggctagcgacttcaacctgcctcccgtggtggctaaggagatcgtggccagctgcgacaagtgccagctgaagggcgaggccatgcacggccaggtggactgcagccccggcatctggcagctggtttgcacccacctggagggcaaggtgatcctggtggccgtgcacgtggcctccggctacatcgaggccgaggtgatccccgccgagaccggccaggagaccgcctacttcctgctgaagctggccggccgctggcccgtgaagaccatccacaccgacaacggcagcaacttcaccagcgccaccgtgaaggctgcctgctggtgggccggcatcaagcaggagttcggcatcccctacaacccccagtctcagggcgtggtggagagcatgaacaaggagctgaagaagatcatcggccaggtgagggaccaggccgagcacctgaagaccgccgtgcagatggccgtgttcatccacaacttcaagaggaagggcggcatcggcggctacagcgccggcgagaggatcgtggacatcatcgccaccgacatccagaccaaggagctgcagaagcagatcaccaagatccagaacttcagggtgtactacagggacagcaggaaccctctgtggaagggccccgccaagctgctgtggaagggcgagggcgccgtggtgatccaggacaacagcgacatcaaggtggtgcccaggaggaaggccaagatcatcagggactacggcaagcagatggccggcgacgactgcgtggcctccaggcaggacgaggactga
9、具體地,所述的含有cas9-6×c/d?box序列的質(zhì)粒的制備包括:將pcmv-gfp質(zhì)粒中的gfp到poly(a)之間的序列替換為atg+cas9序列+taa+6×c/d?box序列。
10、進(jìn)一步地,所述的含有cas9-6×c/d?box序列的質(zhì)粒的制備分兩步進(jìn)行,先插入cas9序列,然后插入6×c/d?box序列,獲得pcmv-cas9-6×c/d?box質(zhì)粒。
11、具體地,所述的目的細(xì)胞選自hek293、hek293-t、hek293-sf、te671中的一種。
12、進(jìn)一步地,所述的目的細(xì)胞為hek293-t。
13、又一方面,本發(fā)明提供了一種利用上述的vlp載體遞送mrna的方法,包括以下步驟:
14、(1)目的細(xì)胞培養(yǎng),換液,孵育;
15、(2)將含有cas9-6×c/d?box序列的質(zhì)粒、含有l(wèi)7ae序列的質(zhì)粒、載有vsvg基因的包裝質(zhì)粒、載有rev基因的包裝輔助質(zhì)粒、plv-egfp-u3-sp.grna質(zhì)粒與載有g(shù)ag和pol基因的包裝輔助質(zhì)粒共轉(zhuǎn)染目的細(xì)胞;
16、(3)離心,棄上清,濃縮,得到含外源目的mrna的vlp顆粒。
17、具體地,步驟(1)中換液為換成2%?fbsdmem;
18、具體地,步驟(1)中目的細(xì)胞培養(yǎng)具體為每孔中加入1.6e5個/cm2?hek-293t細(xì)胞,培養(yǎng)24h。
19、具體地,步驟(1)中所述的目的細(xì)胞選自hek293、hek293-t、hek293-sf、te671中的一種。
20、進(jìn)一步地,步驟(1)中所述的目的細(xì)胞為hek293-t。
21、具體地,步驟(2)的轉(zhuǎn)染過程中按照總dna與pei的質(zhì)量體積比為1:(1-5)的比例進(jìn)行轉(zhuǎn)染。
22、進(jìn)一步地,步驟(2)中總dna與pei的質(zhì)量體積比為1:2。
23、具體地,步驟(2)中轉(zhuǎn)染6h后換液10%?fbsdmem,于72h收集上清。
24、具體地,所述的含有l(wèi)7ae序列的質(zhì)粒的制備包括:將pms2m-ph-gag-pol-d64v質(zhì)粒中ms2c基因替換為l7ae基因,l7ae-ph-gag-pol-d64v質(zhì)粒上l7ae-gag-pol基因序列如seqid?no:3所示。
25、具體地,所述的含有cas9-6×c/d?box序列的質(zhì)粒的制備包括:將pcmv-gfp質(zhì)粒中的gfp到poly(a)之間的序列替換為atg+cas9序列+taa+6×c/d?box序列,得到pcmv-cas9-6×c/d?box質(zhì)粒。
26、具體地,步驟(2)中所述的載有vsvg基因的包裝質(zhì)粒為pmd.2g質(zhì)粒;
27、具體地,步驟(2)中所述的載有rev基因的包裝輔助質(zhì)粒為prsv-rev質(zhì)粒;
28、具體地,步驟(2)中所述的載有g(shù)ag和pol基因的包裝輔助質(zhì)粒為pmdlg/prre-d64v質(zhì)粒。
29、進(jìn)一步地,所述的質(zhì)粒pmd.2g、prsv-rev、pcmv-cas9-6×c/d?box、plv-egfp-u3-sp.grna、l7ae-ph-gag-pol-d64v與pmdlg/prre-d64v的質(zhì)量比為(6-8):(4-9):(20-30):(20-30):(10-15):(10-15)。
30、再進(jìn)一步地,所述的質(zhì)粒pmd.2g、prsv-rev、pcmv-cas9-6×c/d?box、plv-egfp-u3-sp.grna、l7ae-ph-gag-pol-d64v與pmdlg/prre-d64v的質(zhì)量比為7.2:5.77:25:25:12.5:12.5。
31、具體地,所述的pmd.2g和prsv-rev質(zhì)粒來自于第三代慢病毒質(zhì)粒。
32、具體地,所述的plv-egfp-u3-sp.grna的制備包括:向plv慢病毒穿梭質(zhì)粒中插入egfp序列,向u3?region?of?3'ltr中插入序列u6?promoter序列,得到plv-egfp質(zhì)粒,然后將aavs1?grna-f和aavs1?grna-r進(jìn)行退火操作,得到一條aavs1?grna雙鏈,再用aar?i酶對plv-egfp質(zhì)粒進(jìn)行酶切,將aavs1grna雙鏈克隆至plv-egfp質(zhì)粒,得到plv-egfp-u3-aavs1-sp.grna質(zhì)粒。aavs1?grna序列為:ggggccactagggacaggat(seq?id?no:2)。
33、具體地,所述的pmdlg/prre-d64v的制備包括:將pmdlg-prre質(zhì)粒pol區(qū)域的integrase基因的64位氨基酸gat(d)突變?yōu)間tt(v)。
34、具體地,步驟(3)中離心的轉(zhuǎn)速為25000rpm,離心的時間為1.5-2.5h。
35、進(jìn)一步地,步驟(3)中離心的溫度為4℃,離心的時間為2h。
36、具體地,步驟(3)中濃縮具體為棄上清后加入原上清體積1/50的pbs,過夜溶解重懸。
37、又一方面,本發(fā)明提供了上述的vlp載體或上述的遞送mrna的方法在提高vlp載體的mrna遞送效率和cas9或cas9衍生物的編輯效率中的應(yīng)用。
38、本發(fā)明的有益效果為:
39、本發(fā)明通過對蛋白-莖環(huán)結(jié)構(gòu)組合的優(yōu)化,篩選出特異性結(jié)合和遞送效率更高的l7ae-c/d?box組合,提高vlp載體的mrna遞送效率和crispr-cas9編輯效率。