本發(fā)明涉及慢病毒載體，尤其涉及一種基于慢病毒載體系統(tǒng)的hiv-1基因型耐藥檢測(cè)質(zhì)控品及其制備方法和應(yīng)用。

背景技術(shù)：

1、聯(lián)合抗逆轉(zhuǎn)錄病毒治療(combined?antiretroviral?therapy，cart)是目前治療艾滋病最為有效的方法。然而，隨著治療時(shí)間的延長(zhǎng)以及hiv-1本身高度變異和快速?gòu)?fù)制的特性，耐藥問(wèn)題逐漸成為cart過(guò)程中必須面對(duì)的挑戰(zhàn)。為實(shí)現(xiàn)高效的cart，準(zhǔn)確的hiv-1耐藥檢測(cè)至關(guān)重要。hiv-1基因型耐藥檢測(cè)能夠及時(shí)評(píng)估抗病毒治療失敗患者的耐藥狀況，為臨床醫(yī)生調(diào)整用藥方案提供重要的科學(xué)依據(jù)。目前，從事hiv-1基因型耐藥檢測(cè)的實(shí)驗(yàn)室主要采用實(shí)驗(yàn)室自建方法進(jìn)行檢測(cè)。因此，檢測(cè)能力和檢測(cè)質(zhì)量亟需確認(rèn)和監(jiān)管，通過(guò)hiv-1基因型耐藥檢測(cè)質(zhì)控品開(kāi)展的質(zhì)量控制至關(guān)重要。

2、然而，目前多采用含有耐藥突變的hiv-1感染者臨床樣本和耐藥毒株制備耐藥質(zhì)控品，這些方法存在以下局限性：臨床樣本來(lái)源受限，毒株分離培養(yǎng)操作復(fù)雜、技術(shù)難度高，且兩者均存在潛在的生物安全風(fēng)險(xiǎn)。因此，亟需開(kāi)發(fā)一種可持續(xù)大量獲得且安全的hiv-1基因型耐藥檢測(cè)質(zhì)控品，可用于hiv-1耐藥檢測(cè)實(shí)驗(yàn)室的室內(nèi)質(zhì)控和室間質(zhì)量評(píng)價(jià)，發(fā)現(xiàn)并解決檢測(cè)中存在的問(wèn)題，確保檢測(cè)結(jié)果的準(zhǔn)確性和可靠性。

3、慢病毒載體(lentiviral?vectors，lv)是一種基于hiv-1的重組逆轉(zhuǎn)錄病毒載體，已被廣泛用作病毒載體工具?；趌v系統(tǒng)制備的假病毒能夠高度模擬天然病毒的特性，生物安全性高，可在生物安全2級(jí)實(shí)驗(yàn)室中操作，滿足大多數(shù)實(shí)驗(yàn)室的需求。與臨床樣本和耐藥毒株相比，lv系統(tǒng)具有獨(dú)特的基因編輯優(yōu)勢(shì)，能夠通過(guò)在表達(dá)載體質(zhì)粒上設(shè)計(jì)和引入多個(gè)耐藥突變，從而提供更多樣化的hiv-1基因型耐藥檢測(cè)質(zhì)控品。此外，lv系統(tǒng)包裝假病毒的流程簡(jiǎn)便，易于操作?；谏鲜鰞?yōu)勢(shì)，本發(fā)明利用lv系統(tǒng)建立了開(kāi)發(fā)了一種新型的hiv-1基因型耐藥檢測(cè)質(zhì)控品及其制備方法。

技術(shù)實(shí)現(xiàn)思路

1、本發(fā)明的目的在于提供了一種基于慢病毒載體系統(tǒng)的hiv-1基因型耐藥檢測(cè)質(zhì)控品的制備方法。本發(fā)明利用改良后的四質(zhì)粒載體技術(shù)平臺(tái)，成功制備了一種含耐藥突變k103n的新型hiv-1假病毒基因型耐藥檢測(cè)質(zhì)控品。本發(fā)明提供的的hiv-1基因型耐藥檢測(cè)質(zhì)控品具有良好的均一性和穩(wěn)定性；能夠模擬天然病毒特性，實(shí)現(xiàn)對(duì)核酸檢測(cè)全流程的質(zhì)量控制；可大量制備，成本低；可根據(jù)實(shí)際檢測(cè)的質(zhì)控需求通過(guò)基因編輯制備含有特定耐藥突變位點(diǎn)的質(zhì)控品。本發(fā)明提供了一種利用慢病毒載體系統(tǒng)制備hiv-1假病毒基因型耐藥檢測(cè)質(zhì)控品可靠的技術(shù)平臺(tái)。

2、為了實(shí)現(xiàn)上述發(fā)明目的，本發(fā)明提供以下技術(shù)方案：

3、本發(fā)明提供了一種重組載體表達(dá)質(zhì)粒pll3.7-u6-pol，其特征在于，所述重組載體表達(dá)質(zhì)粒的核苷酸序列如seq?id?no.1所示；

4、seq?id?no.1：

5、gtcgacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagcgcgttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccggccgcgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatcggcactgcgtgcgccaattctgcagacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggggatccaccaaatgaaagattgtactgagagacaggccaattttttagggaaaatctggccttcccacaagggaaggccagggaatttccttcagagcagaccagagccaacagccccaccagaggagagcttcaggtttggggaagggacaacaactccatctcagaagcaggagacaatagacaaggaactatatcccttgacttccctcaaatcactctttggcaacgacccctcgtcacaataaagatagaggggcaattaaaggaagctctattagatacaggagcagatgatacagtattagaagaaatgaatttaacagggaaatggaaaccaaaaatgatagggggaattggaggttttatcaaagtaagacagtatgaacaggtacatgtagaaatttgcggacacaaagctataggtacagtattagtgggacctacacctgtcaatataattggaagaaatctattgactcagattggctgcactttaaattttccaatcagtcccattgaaactataccagtaaaattaaagccaggaatggatggcccaaaggttaaacaatggccattgacagaagaaaaaataaaagcattaacagaaatttgtgatgaaatggaaaaggaaggaaaaattacaaaaattgggcctgaaaatccatataacaccccaatatttgctataaaaaagaaggacagtactaagtggagaaaattagtagatttcagggaactcaataaaagaactcaagatttttgggaagttcaattaggaataccacacccagcagggttaaaaaagaacaaatcagtaacagtactggatgtgggggatgcatatttttcagttcctttacatgaagaattcaggaaatatactgcattcaccatacctagtataaacaatgaaacaccagggattaggtatcagtacaatgtacttccacaaggatggaaaggatcgccagcaatattccaaaatagcatgacaaaaatattagagccttttagaaaacaaaatccagacataattatttatcaatacatggatgatttgtatgtaggatctgacttagagataggacaacatagaacaaaaatagaagaactgagacaacatctgttgaggtggggatttaccacaccagacaagaaacatcagaaagaacctccattcctttggatggggtatgaactccatcctgataaatggacagtacaacctatacagctgccagaaaaggatagctggactgtcaatgatatacaaaagctagtgggaaaattaaattgggcaagtcagatttaccctggaattaaggtaaggcaactgtgtaaactccttaggggagtcaaggcactaacagatatagtaacactaactgaagaagcagaattagaattggcagaaaacagggaaattctaaaagaaccagtacatggggcatactatgacccatcaaaagacttgatagctgaaatacagaaacaagggcaagatcaatggacatatcaaatttaccaagaaccattcaaaaatctaaaaacagggaagtatgcaaaaatgaggactgcccacaccaatgatgtaagacaattaacagaggctgtgcagaaaatagccatggaaagcatagtaatatggggaaagactcctaaatttagattacccatccacaaagaaacatgggagacatggtggacagactattggcaagccacctggattcctgagtgggaatttgttaataccccgcccttagtaaaattatggtatcagctagaaaaagatcccatagtaggggcagaaactttctatgtagatggagcagctaatagagagactaaaatgggaaaggcagggtatgttactgacaggggaagaaaaaaagctgtttctctaactgaaacaacaaatcagaagactgaattgcaagcaattgttagctttgcaagattcaggatcagaagtaaacatagtaacagattcacagtatgcattagggattattcaagcacaaccagataaaagtgaatcagagttagttaaccaaataatagaacaactaataaaaaaggaaagagtctacctgtcatgggtaccagcacataaaggaattggaggaaatgaacaagttgataaactagtaagtagtggaatcaggaaagtactatttctagatggaatagacaaagctcaagaagagcatgaaaggtatcacagcaattggagagcaatggctagtgactttaatctaccacctatagtagcaaaagaaatagtggctagctgtgatcaatgtcagctaaaaggagaggccatgcatggacaagtagactgtagcccaggaatatggcaattagattgtacacatttagaaggaaaaatcatcctggtagcagtccatgtagccagtggatacatggaagcagaggttatcccagcagaaacaggacaagagacagcatactttatactaaaattagcaggaagatggccagtcaaagtaatacatacagacaatggtagtaatttcaccagtgctgcagttaaggcggcctgttggtgggcaggtgtccaacaggaatttggaatcccctataatccccaaagtcagggagtagtagaatctatgaataaagaactcgagcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatccgctagcgctaccggtcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaggaattcgtcgagggacctaataacttcgtatagcatacattatacgaagttatacatgtttaagggttccggttccactaggtacaattcgatatcaagcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcatcgataccgtcgacctcgatcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcaggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattggcagaactacacaccagggccagggatcagatatccactgacctttggatggtgctacaagctagtaccagttgagcaagagaaggtagaagaagccaatgaaggagagaacacccgcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaagtattagagtggaggtttgacagccgcctagcatttcatcacatggcccgagagctgcatccggactgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagcatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgac。

6、本發(fā)明還提供了所述的重組載體表達(dá)質(zhì)粒在制備慢病毒載體系統(tǒng)中的應(yīng)用。

7、本發(fā)明還提供了所述重組載體表達(dá)質(zhì)粒的構(gòu)建方法，包括如下步驟：

8、(1)含有耐藥突變k103n的pol區(qū)基因序列2051-4700nt作為目的序列；

9、(2)將目的序列插入載體表達(dá)質(zhì)粒pll3.7-u6-del，得到重組載體表達(dá)質(zhì)粒pll3.7-u6-pol；

10、步驟(1)中所述pol區(qū)2051-4700nt的核苷酸序列如seq?id?no.2所示；

11、seq?id?no.2：

12、accaaatgaaagattgtactgagagacaggccaattttttagggaaaatctggccttcccacaagggaaggccagggaatttccttcagagcagaccagagccaacagccccaccagaggagagcttcaggtttggggaagggacaacaactccatctcagaagcaggagacaatagacaaggaactatatcccttgacttccctcaaatcactctttggcaacgacccctcgtcacaataaagatagaggggcaattaaaggaagctctattagatacaggagcagatgatacagtattagaagaaatgaatttaacagggaaatggaaaccaaaaatgatagggggaattggaggttttatcaaagtaagacagtatgaacaggtacatgtagaaatttgcggacacaaagctataggtacagtattagtgggacctacacctgtcaatataattggaagaaatctattgactcagattggctgcactttaaattttccaatcagtcccattgaaactataccagtaaaattaaagccaggaatggatggcccaaaggttaaacaatggccattgacagaagaaaaaataaaagcattaacagaaatttgtgatgaaatggaaaaggaaggaaaaattacaaaaattgggcctgaaaatccatataacaccccaatatttgctataaaaaagaaggacagtactaagtggagaaaattagtagatttcagggaactcaataaaagaactcaagatttttgggaagttcaattaggaataccacacccagcagggttaaaaaagaacaaatcagtaacagtactggatgtgggggatgcatatttttcagttcctttacatgaagaattcaggaaatatactgcattcaccatacctagtataaacaatgaaacaccagggattaggtatcagtacaatgtacttccacaaggatggaaaggatcgccagcaatattccaaaatagcatgacaaaaatattagagccttttagaaaacaaaatccagacataattatttatcaatacatggatgatttgtatgtaggatctgacttagagataggacaacatagaacaaaaatagaagaactgagacaacatctgttgaggtggggatttaccacaccagacaagaaacatcagaaagaacctccattcctttggatggggtatgaactccatcctgataaatggacagtacaacctatacagctgccagaaaaggatagctggactgtcaatgatatacaaaagctagtgggaaaattaaattgggcaagtcagatttaccctggaattaaggtaaggcaactgtgtaaactccttaggggagtcaaggcactaacagatatagtaacactaactgaagaagcagaattagaattggcagaaaacagggaaattctaaaagaaccagtacatggggcatactatgacccatcaaaagacttgatagctgaaatacagaaacaagggcaagatcaatggacatatcaaatttaccaagaaccattcaaaaatctaaaaacagggaagtatgcaaaaatgaggactgcccacaccaatgatgtaagacaattaacagaggctgtgcagaaaatagccatggaaagcatagtaatatggggaaagactcctaaatttagattacccatccacaaagaaacatgggagacatggtggacagactattggcaagccacctggattcctgagtgggaatttgttaataccccgcccttagtaaaattatggtatcagctagaaaaagatcccatagtaggggcagaaactttctatgtagatggagcagctaatagagagactaaaatgggaaaggcagggtatgttactgacaggggaagaaaaaaagctgtttctctaactgaaacaacaaatcagaagactgaattgcaagcaattgttagctttgcaagattcaggatcagaagtaaacatagtaacagattcacagtatgcattagggattattcaagcacaaccagataaaagtgaatcagagttagttaaccaaataatagaacaactaataaaaaaggaaagagtctacctgtcatgggtaccagcacataaaggaattggaggaaatgaacaagttgataaactagtaagtagtggaatcaggaaagtactatttctagatggaatagacaaagctcaagaagagcatgaaaggtatcacagcaattggagagcaatggctagtgactttaatctaccacctatagtagcaaaagaaatagtggctagctgtgatcaatgtcagctaaaaggagaggccatgcatggacaagtagactgtagcccaggaatatggcaattagattgtacacatttagaaggaaaaatcatcctggtagcagtccatgtagccagtggatacatggaagcagaggttatcccagcagaaacaggacaagagacagcatactttatactaaaattagcaggaagatggccagtcaaagtaatacatacagacaatggtagtaatttcaccagtgctgcagttaaggcggcctgttggtgggcaggtgtccaacaggaatttggaatcccctataatccccaaagtcagggagtagtagaatctatgaataaagaa。

13、步驟(2)中所述插入時(shí)使用pol區(qū)2051-4700nt的上游引物和下游引物；

14、pol區(qū)2051-4700nt的上游引物的核苷酸序列如seq?id?no.3所示；

15、pol區(qū)2051-4700nt的下游引物的核苷酸序列如seq?id?no.4所示；

16、所述載體表達(dá)質(zhì)粒pll3.7-u6-del的核苷酸序列如seq?id?no.5所示；

17、seq?id?no.5：

18、5’-gtcgacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagcgcgttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccggccgcgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatcggcactgcgtgcgccaattctgcagacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggttagtaccgggcccgctctagaaacgcgcggtgaccctcgaggtcgacggtatcgataagctcgcttcacgagattccagcaggtcgagggacctaataacttcgtatagcatacattatacgaagttatattaagggttccaagcttaagcggccgcgtggataaccgtattaccgccatgcattagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatccgctagcgctaccggtcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaggaattcgtcgagggacctaataacttcgtatagcatacattatacgaagttatacatgtttaagggttccggttccactaggtacaattcgatatcaagcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgcctccccgcatcgataccgtcgacctcgatcgagacctagaaaaacatggagcaatcacaagtagcaatacagcagctaccaatgctgattgtgcctggctagaagcacaagaggaggaggaggtgggttttccagtcacacctcaggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattggcagaactacacaccagggccagggatcagatatccactgacctttggatggtgctacaagctagtaccagttgagcaagagaaggtagaagaagccaatgaaggagagaacacccgcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaagtattagagtggaggtttgacagccgcctagcatttcatcacatggcccgagagctgcatccggactgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagcatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgac-3’。

19、本發(fā)明還提供了一種慢病毒載體系統(tǒng)，包括所述的重組載體表達(dá)質(zhì)粒pll3.7-u6-pol、骨架質(zhì)粒prsv-rev和pmdlgag-pol?rre、包膜質(zhì)粒pmd2.g。

20、本發(fā)明還提供了一種利用所述慢病毒載體系統(tǒng)制備hiv-1假病毒的方法，包括如下步驟：

21、(1)利用所述的hiv-1慢病毒載體系統(tǒng)轉(zhuǎn)染hek293t細(xì)胞，質(zhì)粒與轉(zhuǎn)染試劑混合靜置后緩緩加入培養(yǎng)基中；

22、(2)轉(zhuǎn)染后，培養(yǎng)44～52h，收集培養(yǎng)上清液；

23、(3)將培養(yǎng)上清液離心后過(guò)濾，得到所述的hiv-1假病毒上清液；

24、步驟(1)中所述轉(zhuǎn)染時(shí)采用的轉(zhuǎn)染體系為：7～9μg重組載體表達(dá)質(zhì)粒、3～5μgprsv-rev、3～5μgpmdlgag-pol?rre和3～5μg?pmd2.g，46～50μl?lipofectamine?2000添加至240～260μl?optimem(gibco)中混勻，轉(zhuǎn)染0.8～1.2×107cells/t75的293t細(xì)胞；

25、步驟(2)中所述培養(yǎng)的時(shí)間為44～52h；

26、步驟(3)中所述離心的轉(zhuǎn)速為1800～2200rpm，離心的時(shí)間為8～12min，所述過(guò)濾時(shí)采用0.45μm微孔過(guò)濾器。

27、本發(fā)明還提供了所述的方法制備得到的hiv-1假病毒。

28、本發(fā)明還提供了所述的hiv-1假病毒在制備hiv-1基因型耐藥檢測(cè)質(zhì)控品的應(yīng)用。

29、本發(fā)明的上述技術(shù)方案的有益效果如下：

30、(1)生物安全性高

31、本發(fā)明通過(guò)四質(zhì)粒慢病毒載體系統(tǒng)，有效防止了因攜帶hiv-1rna片段而導(dǎo)致的假病毒“感染性重現(xiàn)”，具有高度生物安全性。實(shí)施例數(shù)據(jù)表明，本發(fā)明制備hiv-1基因型耐藥檢測(cè)質(zhì)控品所使用的hiv-1假病毒僅具有單輪感染能力。與臨床樣本和耐藥毒株相比，假病毒的高生物安全性有效克服了臨床樣本和耐藥毒株存在生物安全風(fēng)險(xiǎn)的問(wèn)題，可以在生物安全2級(jí)實(shí)驗(yàn)室進(jìn)行操作，降低對(duì)操作環(huán)境以及后續(xù)的運(yùn)輸條件與儲(chǔ)存環(huán)境的生物安全等級(jí)要求，能夠滿足大多數(shù)實(shí)驗(yàn)室的實(shí)驗(yàn)需求，方便操作、運(yùn)輸和儲(chǔ)存。

32、(2)高度模擬天然病毒的生物學(xué)特性

33、慢病毒載體系統(tǒng)包裝的假病毒能夠高度模擬天然病毒的特性，使其與臨床樣本和耐藥毒株一樣，能夠模擬臨床樣本的檢測(cè)流程，可對(duì)耐藥檢測(cè)有效地進(jìn)行全流程質(zhì)量控制，確保檢測(cè)結(jié)果的準(zhǔn)確性和可靠性。

34、(3)制備流程簡(jiǎn)便，易于持續(xù)大量獲得

35、基于本發(fā)明所提供的基于慢病毒載體系統(tǒng)的假病毒包裝過(guò)程簡(jiǎn)單，且載體表達(dá)質(zhì)粒pll3.7-u6-pol上包含輔助病毒dna核內(nèi)運(yùn)輸?shù)膆iv-1pol基因的cppt/cts序列，轉(zhuǎn)染效率高和包裝產(chǎn)物滴度高。實(shí)施例數(shù)據(jù)表明，本發(fā)明hiv-1假病毒包裝方法可獲得高達(dá)到109copies/ml的hiv-1假病毒上清液。與臨床樣本和耐藥毒株相比，假病毒更容易制備與持續(xù)大量獲得，且易于儲(chǔ)存與運(yùn)輸。

36、(4)具備良好質(zhì)控品性能

37、實(shí)施例數(shù)據(jù)表明，本發(fā)明所制備的質(zhì)控品均一性良好；在室溫(25℃)、4℃和-20℃條件下儲(chǔ)存28天，仍可擴(kuò)增出含有耐藥突變的目的序列，具有較好的穩(wěn)定性。因此，本發(fā)明提供的hiv-1基因型耐藥檢測(cè)質(zhì)控品具有良好的應(yīng)用價(jià)值。

38、(5)具有特有的基因編輯優(yōu)勢(shì)

39、與臨床樣本和耐藥毒株只能攜帶特定耐藥突變不同，本發(fā)明通過(guò)慢病毒載體系統(tǒng)制備耐藥檢測(cè)質(zhì)控品的方法具有獨(dú)特的基因編輯優(yōu)勢(shì)。本發(fā)明成功插入了2648bp的外源基因，已有研究表明慢病毒載體系統(tǒng)可以攜帶8kb左右的外源基因?；诒景l(fā)明提供的方法未來(lái)可將多種常見(jiàn)耐藥突變同時(shí)包入假病毒顆粒中，從而制備符合實(shí)際需求的hiv-1假病毒基因型耐藥檢測(cè)質(zhì)控品，為耐藥檢測(cè)質(zhì)控工作提供了多樣化的質(zhì)控品選擇。