WO2022160562A1 - 一种慢病毒滴度检测引物、试剂盒及检测方法 - Google Patents

一种慢病毒滴度检测引物、试剂盒及检测方法 Download PDF

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WO2022160562A1
WO2022160562A1 PCT/CN2021/100013 CN2021100013W WO2022160562A1 WO 2022160562 A1 WO2022160562 A1 WO 2022160562A1 CN 2021100013 W CN2021100013 W CN 2021100013W WO 2022160562 A1 WO2022160562 A1 WO 2022160562A1
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primer
sequence
seq
lentivirus
kit
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施金秀
罗燕
蔡育宜
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云舟生物科技（广州）有限公司
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Definitions

  • the invention relates to the field of virus molecular biology, in particular to a lentivirus titer detection primer, a kit and a detection method.
  • Lentiviral vector is a retroviral vector derived from human immunodeficiency virus type I (Human immunodeficiency virus, HIV).
  • human immunodeficiency virus, HIV Human immunodeficiency virus, HIV.
  • the advantages of dividing and non-dividing cells have been widely used in basic research and clinical gene therapy.
  • the random integration of lentivirus into the host genome can induce up-regulation of gene expression near the integration site, and even activate proto-oncogenes, leading to tumorigenesis.
  • the third-generation self-inactivating (SIN) lentiviral vector system has been developed.
  • the SIN vector Compared with the second-generation lentiviral vector, the SIN vector has the following characteristics: 1) Deletion of 5'LTR and 3 U3 region with promoter/enhancer activity in 'LTR, and replace U3 region of 5'LTR with RSV promoter to compensate for its transcriptional activity; 2) delete the tat gene necessary for virus replication; 3) replace the rev gene Expressed on a separate vector.
  • This not only further improves the biosafety of lentiviruses and reduces the possibility of replicating lentiviruses (RCL), but also greatly expands the application scope of lentiviral vectors in the field of gene therapy. Lentiviral vector-based gene therapy has become an ideal choice for a variety of genetic diseases.
  • the first lentiviral vector-based chimeric antigen receptor T cell (CAR-T) therapy was approved by the FDA in August 2017 , for the treatment of acute lymphoblastic leukemia in children and adults.
  • CAR-T chimeric antigen receptor T cell
  • lentiviral titers are prerequisite for accurate dosing and patient safety. Therefore, how to accurately measure lentiviral titers is crucial.
  • lentiviral titer determination there are three main methods for lentiviral titer determination, namely, ELISA to detect capsid protein p24, RT-qPCR to detect lentiviral genome copy number, and qPCR to detect lentiviral functional titer.
  • the ELISA method determines the number of virus particles by measuring the amount of p24 protein.
  • the titer data obtained by the detection not only includes the expected infectious activity of lentivirus particles, but also includes free p24 protein and genome-defective virus.
  • the virus titer measured by ELISA method is often much higher than the actual virus titer.
  • RT-qPCR method detects the copy number of lentiviral genome cDNA by reverse transcribing the lentiviral RNA genome into cDNA, and then using it as a template qPCR.
  • the RT-qPCR method avoids the interference of the empty shell virus on the titer detection, the existence of the genome-defective virus still has a huge impact on the titer detection results, which is as much as 20 times higher than the actual virus titer. And the fluctuation range between different groups is very large.
  • the qPCR method uses the cell genome extracted from the virus transduced cells as a template for qPCR, and the obtained data can directly reflect the copy number of the viral genome integrated into the host genome. Compared with the above two methods, the qPCR method can better reflect the lentivirus. Actual functional titers.
  • upstream and downstream primer design strategies involved in the detection of lentivirus titer by qPCR: 1) upstream and downstream primers are designed on the virus-specific sequences between the two LTRs such as gag and env; 2) upstream primers are designed on In the U5 region, the downstream primer is designed on the virus-specific sequence; 3) The upstream primer is designed in the R region, and the downstream primer is designed in the U5 region.
  • DNase I or benzonase is often added to the virus solution for digestion.
  • the above three primers can accurately determine the number of copies in the genome after lentiviral transduction, that is, to obtain the accurate functional titer of the lentivirus.
  • DNase I or benzonase is added to digest the residual plasmid DNA during virus purification, a small amount of DNA remains unavoidable. At this time, it is obviously inappropriate to use the above primers to detect virus titer.
  • the primers of the existing qPCR-based lentiviral functional titer assay method are designed on the lentiviral backbone.
  • the copy number detected by this method is the difference between the residual plasmid DNA copy number and the The sum of viral integrated copy numbers cannot accurately reflect the true functional titer and integrated copy number of lentivirus.
  • the first object of the present invention is to provide a primer in order to eliminate the influence of residual plasmid DNA.
  • the second object of the present invention is to provide a kit.
  • the third object of the present invention is to provide a lentivirus titer detection method.
  • the first aspect of the present invention provides a primer, based on the principle that the U3 region in the 3'LTR of the lentivirus will be copied to the 5'LTR during the reverse transcription process, and the form of the previous virus will be integrated into the genome.
  • the upstream primer U3-F was designed for the residual U3 region in the 3'LTR of the SIN vector, and the downstream primer LV-R was designed on the lentiviral backbone downstream of the 5'LTR.
  • primers are used to amplify a fragment whose sequence is as shown in SEQ ID NO.1, or a sequence fragment whose sequence is complementary to that shown in SEQ ID NO.1, or a sequence that includes a sequence as shown in SEQ ID NO.1
  • a sequence or its complement has a sequence fragment in which one or more bases have been deleted, substituted or inserted.
  • sequence of the primer is:
  • U3-F AGGGCTAATTCACTCCCAACG (SEQ ID NO. 2);
  • LV-R CGCTTTCAAGTCCCTGTTCG (SEQ ID NO. 3).
  • the forward primer RSV-F can be designed on the RSV promoter upstream of the 5'LTR of the lentiviral plasmid, and the downstream primer is still LV-R, wherein the sequence of RSV-F is is AACAGACGGGTCTGACATGGAT (SEQ ID NO: 4).
  • the second aspect of the present invention provides a kit comprising the primers described in the first aspect of the present invention.
  • kit also includes an internal reference primer.
  • sequence of the internal reference primer is:
  • BMP2-F TAGGGTAGACAGAGCCAAGG (SEQ ID NO. 5);
  • BMP2-R AGCACAGGACAAGAAAGTCATTG (SEQ ID NO. 6).
  • the detection reagent includes a standard of the sample to be tested.
  • the detection reagent comprises a standard of an internal reference fragment.
  • a third aspect of the present invention provides a lentivirus titer detection method, comprising the following steps:
  • S1 cell counting and transduction of lentivirus into cells
  • S3 Use the primers of the first aspect of the present invention or the kit of the second aspect of the present invention to perform qPCR to detect the lentivirus titer.
  • the present invention is based on the principle that the U3 region in the 3'LTR of the lentivirus will be copied to the 5'LTR during the reverse transcription process, and the form of the provirus (provirus) is integrated into the genome, in order to obtain the accurate integrated copy number of the lentivirus.
  • the upstream primer U3-F for the residual U3 region in the 3'LTR of the SIN vector
  • the downstream primer LV-R was designed on the lentiviral backbone downstream of the 5'LTR.
  • BMP2 standard and the test sample related to the genome copy number Sample standard 5' LTR.
  • the forward primer RSV-F can be designed on the RSV promoter upstream of the 5'LTR of the lentiviral plasmid, and the downstream primer is still LV-R.
  • the U3-F and LV-R primer pairs could only detect the copy number of the lentiviral genome integrated into the host genome, thus eliminating the interference of residual plasmid DNA, and finally established a An efficient, accurate and reproducible lentiviral functional titer determination method can accurately confirm the integrated copy number of lentiviral vectors.
  • FIG. 1 is a schematic diagram of the distribution of qPCR primers in Example 1 of the present invention.
  • Fig. 2 is the sequencing peak map of pMD TM 18-T-BMP2-gag in Example 2 of the present invention
  • Figure 3 is the dissolution curve in Example 3 of the present invention.
  • the present invention is based on the principle that the U3 region in the 3'LTR of the lentivirus will be copied to the 5'LTR during the reverse transcription process, and the form of the provirus (provirus) is integrated into the genome, in order to obtain the accurate integrated copy number of the lentivirus.
  • the upstream primer U3-F is designed for the residual U3 region in the 3'LTR of the SIN vector
  • the downstream primer LV-R is designed on the lentiviral backbone downstream of the 5'LTR.
  • the BMP2 standard and the test sample related to the genome copy number are set Standard 5' LTR.
  • the forward primer RSV-F can be designed on the RSV promoter upstream of the 5'LTR of the lentiviral plasmid, and the downstream primer is still LV-R.
  • the U3-F and LV-R primer pairs could only detect the copy number of the lentiviral genome integrated into the host genome, thus eliminating the interference of residual plasmid DNA, and finally established a An efficient, accurate and reproducible lentiviral functional titer determination method can accurately confirm the integrated copy number of lentiviral vectors.
  • the amplified fragment length was 258 bp.
  • the resulting primer sequences are:
  • U3-F AGGGCTAATTCACTCCCAACG (SEQ ID NO. 2);
  • LV-R CGCTTTCAAGTCCCTGTTCG (SEQ ID NO. 3).
  • the forward primer RSV-F can be designed on the RSV promoter upstream of the 5'LTR of the lentiviral plasmid, and the downstream primer is still LV-R, where the sequence of RSV-F is AACAGACGGGTCTGACATGGAT ( SEQ ID NO. 4).
  • the specific primer distribution is shown in Figure 1.
  • BMP2-F TAGGGTAGACAGAGCCAAGG (SEQ ID NO. 5);
  • BMP2-R AGCACAGGACAAGAAAGTCATTG (SEQ ID NO. 6).
  • the amplified sequence is shown in SEQ ID NO.7: TAGGGTAGACAGAGCCAAGGGCAGAGTTTTCAGAGA TAGTAT TGAAAA ATCAAA GCCCAGGGCCCCAAAGTCTTTCTAATTTATAGTTGATCTGGGCCTGGTTTGGAAGATTTTGAATCCCAATCTAATCCCCGTGGGAGATCAATACTACAATCAATCTTATTGTTTCCACAATGACTTTCTTGTCCTGTGCT (SEQ ID NO.7).
  • the amplified length was 194 bp.
  • a kit for lentivirus titer detection comprising the primers and internal reference primers described in Example 1, as well as a standard substance of a sample to be tested and a standard substance of an internal reference fragment.
  • This standard contains both BMP2 and U3 fragments, so this standard can be used as the standard for the test sample and the internal reference fragment.
  • the 5'LTR of the provirus and part of its downstream sequence were amplified by PCR.
  • the 5' end of the primer carried a homology arm for Gibson reaction, and the PCR product ran 2% gel electrophoresis was performed, the gel was cut and recovered, and the concentration of the recovered product was determined.
  • the PCR amplification system is shown in Table 1, and the primer information is shown in Table 2:
  • SCR-F TGATGCACACAATAGAGGGTTGCT (SEQ ID NO. 10);
  • SCR-R GCAGTGAGCGCAACGCAATT (SEQ ID NO. 11).
  • the average final concentration of the standard fragment prepared in this experiment was 34ng/ ⁇ L, and the fragment size was 2242bp. 5 ⁇ L of this fragment was serially diluted to prepare a standard containing 10 8 copies/ ⁇ L.
  • Embodiment 3 A kind of lentivirus titer detection method
  • gag upstream and downstream primers are designed on the gag gene, which is a virus-specific sequence.
  • the amplified fragment sequence is as follows:
  • the qPCR reaction was carried out according to the standard operating procedures: pre-denaturation at 95°C for 60s, denaturation at 95°C for 15s, annealing at 60°C for 60s, 40 cycles.
  • the genomic copy number of 5'LTR in the same sample divided by the genomic copy number of BMP2 is the MOI of the sample when transduced.
  • the cell count before transduction is 10 6
  • 50 ⁇ L of virus solution diluted 100 times is added during transduction.
  • the genome extracted from lentivirus-transduced 293T cells was used as a template, and the amplification and dissolution curves of the U3-F and LV-R primer pairs were shown in Figure 2. It can be seen from Figure 2 that the Tm value is 85 °C and is a single peak, indicating that the Primer pairs have good specificity.
  • the template was the genomes extracted from 2-well 293T cells transduced with the same amount of virus, named LV-1 and LV-2 respectively, and the initial copy number of the standard was 10 8 , and then serially diluted to 10 7 , 10 6 , and 10 . 5 and 10 4 , the three pairs of primers in Table 6 were used for qPCR detection, and the results are shown in Table 7.
  • the difference between the molecular copy number and CT value between samples LV-1 and LV-2 is relatively small.
  • the data amplified by the gag primer pair reflects the sum of the integrated copy number of lentivirus and the residual viral vector in the genome
  • BMP2 represents the copy number of the genome
  • U3-LV only reflects the number of lentivirus in the genome.
  • the number of copies of viral integration It can be seen from Table 7 that the copy number detected by the U3-LV primer pair in both samples is lower than that detected by gag.
  • the LV-1 and LV-2 samples in The integrated copy number of lentivirus was 0.51 and 0.35, respectively, while if calculated based on the ratio of gag to BMP2, the integrated copy number of lentivirus was inflated to 1.27 and 0.76.

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Abstract

提供了一种慢病毒滴度检测引物、试剂盒及检测方法，该检测试剂盒含有SIN载体上特异性扩增引物，该引物仅能够检测出整合到宿主基因组中的慢病毒基因组拷贝数，从而消除了残留质粒DNA的干扰。

Description

一种慢病毒滴度检测引物、试剂盒及检测方法 技术领域
本发明涉及病毒分子生物学领域，具体为一种慢病毒滴度检测引物、试剂盒及检测方法。
背景技术
慢病毒载体是一种来源于I型人类免疫缺陷病毒(Human immunodeficiency virus,HIV)的逆转录病毒载体，具有宿主范围广、免疫原性低、转染效率高、目的基因稳定高效表达、可感染分裂和非分裂细胞等优点，已被广泛应用于基础研究和临床基因治疗中。慢病毒在宿主基因组中的随机整合可诱发整合位点附近基因表达上调，甚至激活原癌基因，导致肿瘤的发生。为了解决这个潜在的安全问题，目前已开发出第三代自失活(SIN)慢病毒载体系统，与第二代慢病毒载体相比，SIN载体具有以下特点：1)缺失5’LTR及3’LTR中具有启动子/增强子活性的U3区，并将5’LTR的U3区替换为RSV启动子，以补偿其转录活性；2)删除病毒复制所必需的tat基因；3)将rev基因在单独的载体上表达。这不仅进一步提高了慢病毒的生物安全性，降低了复制型慢病毒(RCL)产生的可能性，而且大大扩展了慢病毒载体在基因治疗领域的应用范围。基于慢病毒载体的基因治疗已成为当前多种遗传性疾病的理想选择，第一种基于慢病毒载体的嵌合抗原受体T细胞(CAR-T)疗法tisagenlecleucel已于2017年8月获得FDA批准，用于治疗儿童和成人急性淋巴细胞白血病。慢病毒载体作为一种基因治疗载体，已越来越受到研究者的追捧。
获得准确的慢病毒滴度是实现精确给药剂量和保障患者安全的前提条件，因此，如何精确测定慢病毒滴度便显得至关重要。目前，慢病毒滴度测定的方法主要有三种，分别是ELISA法检测衣壳蛋白p24、RT-qPCR检测慢病毒基因组拷贝数和qPCR检测慢病毒功能滴度。其中，ELISA法通过测定p24蛋白的量来确定病毒颗粒的数量，检测所得到的滴度数据不仅包括了预期的具有感染活性的慢病毒颗粒，还囊括了游离的p24蛋白及基因组缺陷病毒，因此，采用ELISA法所测得的病毒滴度往往远高于实际的病毒滴度。RT-qPCR法通过将慢病毒RNA基因组逆转录成cDNA，然后以之为模板qPCR检测慢病毒基因组cDNA的拷贝数。与ELISA法相比，RT-qPCR法虽然避免了空壳病毒对滴度检测的干扰，但基因组缺陷病毒的存在仍对滴度检测结果造成了巨大影响，比实际病毒滴度高达20倍之多，且不同组间的波动幅度非常大。qPCR法则是以病毒转导细胞后提取的细胞基因组为模板进行qPCR，所得数据能够直观体现病毒基因组整合到宿主基因组中的拷贝数，与上述两种方法相比，qPCR法更能够体现慢 病毒的实际功能滴度。
目前，qPCR法检测慢病毒滴度所涉及的上下游引物设计策略主要有三种：1)上下游引物设计在gag、env等两个LTR之间的病毒特异性序列上；2)上游引物设计在U5区，下游引物设计在病毒特异性序列上；3)上游引物设计在R区，下游引物设计在U5区。在慢病毒的纯化过程中，为了去除残留的质粒DNA污染，往往会在病毒液中加入DNase I或benzonase进行消化。在无慢病毒质粒DNA污染的情况下，上述三种引物都能够准确测定出慢病毒转导后在基因组中的拷贝数，即得到慢病毒的准确功能滴度。然而，即便在病毒纯化过程中加入了DNase I或benzonase以消化残留的质粒DNA，少量的DNA残留仍不可避免，此时用上述引物来检测病毒滴度显然是不合适的。现有的基于qPCR的慢病毒功能滴度测定方法的引物设计在慢病毒骨架上，当转导细胞的病毒中含有病毒质粒DNA残留时，该方法检测出来的拷贝数是残留质粒DNA拷贝数与病毒整合拷贝数之和，并不能准确反映慢病毒的真实功能滴度和整合拷贝数。
发明内容
本发明的第一个目的在于，为了消除残留质粒DNA的影响，提供一种引物。
本发明的第二个目的在于提供一种试剂盒。
本发明的第三个目的在于提供一种慢病毒滴度检测方法。
本发明所采取的技术方案是：
本发明的第一个方面，提供一种引物，基于慢病毒在逆转录过程中会将3’LTR中的U3区复制到5’LTR上，以前病毒的形式整合到基因组中的原理，为了能够得到慢病毒的准确整合拷贝数，针对SIN载体3’LTR中残留的U3区设计上游引物U3-F，下游引物LV-R设计在5’LTR下游的慢病毒骨架上。
进一步地，所述引物用于扩增序列如SEQ ID NO.1所示的片段、或包括序列与SEQ ID NO.1所示序列互补的序列片段、或包括序列与SEQ ID NO.1所示序列或其互补序列具有一个或多个碱基缺失、取代或插入的序列片段。
优选地，所述引物的序列为：
U3-F：AGGGCTAATTCACTCCCAACG(SEQ ID NO.2)；
LV-R：CGCTTTCAAGTCCCTGTTCG(SEQ ID NO.3)。
进一步地，提供上述引物在慢病毒滴度检测中的应用。
进一步地，如需对质粒DNA残留进行检测时，则可在慢病毒质粒5’LTR上游的RSV启动子上设计正向引物RSV-F，下游引物仍为LV-R，其中RSV-F的序列为 AACAGACGGGTCTGACATGGAT(SEQ ID NO：4)。
本发明的第二个方面，提供一种试剂盒，所述试剂盒包含本发明第一方面所述引物。
进一步地，所述试剂盒还包含内参引物。
优选地，所述内参引物的序列为：
BMP2-F：TAGGGTAGACAGAGCCAAGG(SEQ ID NO.5)；
BMP2-R：AGCACAGGACAAGAAAGTCATTG(SEQ ID NO.6)。
进一步地，所述检测试剂包含待测样品的标准品。
进一步地，所述检测试剂包含内参片段的标准品。
本发明的第三个方面，提供一种慢病毒滴度检测方法，包括如下步骤：
S1：细胞计数并将慢病毒转导至细胞；
S2：提取细胞DNA；
S3：用本发明第一方面所述引物或本发明第二方面所述试剂盒进行qPCR检测慢病毒滴度。
本发明的有益效果是：
本发明基于慢病毒在逆转录过程中会将3’LTR中的U3区复制到5’LTR上，以前病毒(provirus)的形式整合到基因组中的原理，为了能够得到慢病毒的准确整合拷贝数，我们针对SIN载体3’LTR中残留的U3区设计上游引物U3-F，下游引物LV-R设计在5’LTR下游的慢病毒骨架上，同时设置基因组拷贝数相关的BMP2标准品和待测样品标准品5’LTR。如需对质粒DNA残留进行检测时，则可在慢病毒质粒5’LTR上游的RSV启动子上设计正向引物RSV-F，下游引物仍为LV-R。当以残留质粒DNA的宿主基因组为模板时，利用U3-F和LV-R引物对仅能够检测出整合到宿主基因组中的慢病毒基因组拷贝数，从而消除了残留质粒DNA的干扰，最终建立了一种高效、准确、重复性好的慢病毒功能滴度测定方法，准确地确认慢病毒载体的整合拷贝数。
附图说明
图1为本发明实施例1中的qPCR引物分布示意图。
图2为本发明实施例2中的pMD TM18-T-BMP2-gag测序峰图
图3为本发明实施例3中的溶解曲线。
具体实施方式
下面结合具体实施例与附图进一步说明本发明的技术方案。下述实施例仅用于示例性说明，不能理解为对本发明的限制。除非特别说明，下述实施例中使用的试剂原料为常规市购 或商业途径获得的试剂原料。除非特别说明，下述实施例中使用的系统为本领域常规使用的设备。
本发明基于慢病毒在逆转录过程中会将3’LTR中的U3区复制到5’LTR上，以前病毒(provirus)的形式整合到基因组中的原理，为了能够得到慢病毒的准确整合拷贝数，针对SIN载体3’LTR中残留的U3区设计上游引物U3-F，下游引物LV-R设计在5’LTR下游的慢病毒骨架上，同时设置基因组拷贝数相关的BMP2标准品和待测样品标准品5’LTR。如需对质粒DNA残留进行检测时，则可在慢病毒质粒5’LTR上游的RSV启动子上设计正向引物RSV-F，下游引物仍为LV-R。当以残留质粒DNA的宿主基因组为模板时，利用U3-F和LV-R引物对仅能够检测出整合到宿主基因组中的慢病毒基因组拷贝数，从而消除了残留质粒DNA的干扰，最终建立了一种高效、准确、重复性好的慢病毒功能滴度测定方法，准确地确认慢病毒载体的整合拷贝数。
实施例1 引物设计
(1)基于慢病毒在逆转录过程中会将3’LTR中的U3区复制到5’LTR上，以前病毒(provirus)的形式整合到基因组中的原理，为了能够得到慢病毒的准确整合拷贝数，针对SIN载体3’LTR中残留的U3区设计上游引物U3-F，下游引物LV-R设计在5’LTR下游的慢病毒骨架上。
所述引物用于扩增目的病毒片段序列如SEQ ID NO.1所示：AGGGCTAATTCACTCCCAACGAAG ACAAGA TCTGCT TTTTGC TTGTAC TGGGTC TCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGG CGCCCGAACAGGGACTTGAAAGCG(SEQ ID NO.1)
片段扩增长度为258bp。
所得引物序列为：
U3-F：AGGGCTAATTCACTCCCAACG(SEQ ID NO.2)；
LV-R：CGCTTTCAAGTCCCTGTTCG(SEQ ID NO.3)。
如需对质粒DNA残留进行检测时，则可在慢病毒质粒5’LTR上游的RSV启动子上设计正向引物RSV-F，下游引物仍为LV-R，其中RSV-F的序列为AACAGACGGGTCTGACATGGAT(SEQ ID NO.4)。具体的引物分布见图1。
(2)内参BMP2(Homo sapiens bone morphogenetic protein 2，骨形态发生蛋白2)引物：
BMP2-F：TAGGGTAGACAGAGCCAAGG(SEQ ID NO.5)；
BMP2-R：AGCACAGGACAAGAAAGTCATTG(SEQ ID NO.6)。
扩增序列如SEQ ID NO.7所示：TAGGGTAGACAGAGCCAAGGGCAGAGTTTTCAGAGA TAGTAT TGAAAA ATCAAA GCCCAGGGCCCCAAAGTCTTTCTAATTTATAGTTGATCTGGGCCTGGTTTGGAAGATTTTGAATCCCAATCTAATCCCCGTGGGAGATCAATACTACAATCAATCTTATTGTTTCCACAATGACTTTCTTGTCCTGTGCT(SEQ ID NO.7)。
扩增长度为194bp。
实施例2 一种用于慢病毒滴度检测试剂盒的制备
一种用于慢病毒滴度检测试剂盒，包含实施例1中所述引物和内参引物，还包含待测样品的标准品、内参片段的标准品。该标准品同时包含BMP2和U3片段，因此可用该标准品作为待测样品和内参片段的标准品。
其中标准品的制备步骤如下所述：
1.PCR扩增provirus的5’LTR序列
以转染慢病毒的细胞基因组DNA为模板，PCR扩增provirus的5’LTR及部分下游序列(5’LTR-LV)，同时引物5’端携带用于Gibson反应的同源臂，PCR产物跑2％凝胶电泳，切胶回收，测定回收产物的浓度。PCR扩增体系见表1，引物信息见表2：
表1 PCR扩增体系
Figure PCTCN2021100013-appb-000001
表2 PCR扩增引物信息
Figure PCTCN2021100013-appb-000002
2.Gibson克隆构建pMD TM18-T-BMP2-gag-5’LTR
2.1 EcoRI酶切已有的pMD TM18-T-BMP2-ENV2质粒，酶切体系及反应条件见表3，37℃反应2～3h：
表3 酶切体系
pMD TM18-T-BMP2-gag 2μg
10×NEBuffer 2.1 2μL
EcoRI 1μL
ddH 2O 添加至20μL
2.2乙醇沉淀回收骨架载体；
2.3将线性化骨架与PCR产物进行Gibson反应，反应体系及条件见表4，50℃反应15min：
表4 Gibson反应体系
Figure PCTCN2021100013-appb-000003
2.4取5μl连接产物转化100μL VB UltraStable感受态细胞；
2.5菌落PCR鉴定LB板上的克隆，鉴定引物如下：
SCR-F：TGATGCACACAATAGAGGGTTGCT(SEQ ID NO.10)；
SCR-R：GCAGTGAGCGCAACGCAATT(SEQ ID NO.11)。
2.6将PCR鉴定阳性克隆酶切并测序，其中BMP2-gag-5’LTR片段的测序峰图见图2，验证了序列正确性。
2.7制备一种通用型的标准品，该标准品含有5’LTR-LV、BMP2和gag三种片段：使用Ahd I与Nde I双酶切上述标准品质粒，跑胶并回收相应条带，使用Qubit荧光计测浓度，测3次求平均值作为最终浓度；
2.8标准品稀释：根据浓度、片段大小、平均每对碱基分子量(660)计算分子数，稀释成10 8标准品。计算方法为：X＝A×6.02×10 23×10 -9/(660×B×10 8)。其中X为需要稀释的倍数，A为片段所测的浓度(单位ng/μL)，B为片段大小(碱基对)。
本次实验制备的标准品片段平均终浓度为34ng/μL，片段大小为2242bp。取5μL该片段逐级稀释制备成含有10 8拷贝数/μL的标准品。
2.9标准品稀释后按10μL一管分装，保存在-80℃冰箱。
实施例3 一种慢病毒滴度检测方法
S1：细胞计数并将慢病毒转导至细胞
1)转导前一天，将293T细胞按3×10 5每孔接种至6孔板中，每孔的液体总体积为2mL，按照每个病毒样品转导两个复孔计算所需接种的数量；
2)转导前先消化一个孔的细胞进行细胞计数，计数结果记为M。取10μL慢病毒样品用培养基稀释至1mL，取50μL稀释后的病毒液加入到293T细胞中，加入10μL 0.5mg/mL Polybrene，轻轻摇晃均匀后放回37℃培养箱继续培养48小时。同时应留一个孔不加任何病毒，作为空白对照。
S2：提取细胞DNA：把6孔板里的细胞分别消化下来，PBS洗一遍，用血液组织细胞基因组提取试剂盒提取细胞样品的基因组DNA(gDNA)，最终用50μL无菌水洗脱。
S3：qPCR检测慢病毒滴度
1)取10 8的qPCR标准品(分别为BMP2片段和5’LTR片段)，用DEPC水逐级稀释至10 7、10 6、10 5、10 4、10 3；
2)按表5配制qPCR反应体系，
表5 qPCR反应体系
Figure PCTCN2021100013-appb-000004
qPCR所用引物见表6：
表6 qPCR引物信息
Figure PCTCN2021100013-appb-000005
其中gag上下游引物设计在gag基因上，是病毒特异性序列。扩增后的片段序列如下：
Figure PCTCN2021100013-appb-000006
3)按照标准操作规程进行qPCR反应：95℃预变性60s，95℃变性15s，60℃退火60s，40个循环。
4)滴度计算
同一样品的5’LTR的基因组拷贝数除以BMP2的基因组拷贝数则为该样品转导时的MOI。滴度计算公式为：滴度(TU/mL)＝1000×MOI×M(细胞计数值)×稀释倍数/转导体积。例如样品的MOI＝1431.915039/7864.708984＝0.182，转导前细胞计数为10 6，转导时加入稀释100倍的病毒液50μL，BMP2和5’LTR的扩增效率一致(接近100％且相差小于5％)，则其滴度为1000×0.182×8×10 5×100×1/50＝3.64×10 8TU/mL。
其中，以慢病毒转导293T细胞提取的基因组为模板，U3-F和LV-R引物对的扩增溶解曲线见图2，从图2可知Tm值为85℃，且为单峰，说明该引物对具有较好的特异性。
模板为相同病毒量转导2孔293T细胞后所提取的基因组，分别命名为LV-1和LV-2，标准品起始拷贝数为10 8，再依次梯度稀释到10 7、10 6、10 5、10 4，利用表6的三对引物进行qPCR检测，结果如表7所示。
表7 qPCR检测结果
Figure PCTCN2021100013-appb-000007
从表7中的三组数据来看，样品LV-1和LV-2之间分子拷贝数及CT值之间差距较小。从不同数据的代表含义来看，gag引物对扩增的数据反映了基因组中慢病毒整合拷贝数和残留病毒载体之和，BMP2代表了基因组的拷贝数，U3-LV则仅反映了基因组中慢病毒整合的拷贝数。由表7可看出，U3-LV引物对在两个样品中测出的拷贝数均比gag测出的要低，根据U3-LV与BMP2的比值可知，LV-1和LV-2样品中的慢病毒整合拷贝数分别为0.51和0.35，而如果根据gag与BMP2的比值计算，慢病毒的整合拷贝数则虚高至1.27和0.76。这充分说明了U3-F和LV-R引物对在慢病毒功能滴度测定方面的有效性，能有效排除残留病毒载体的污染背景。
以上实施例仅为介绍本发明的优选案例，对于本领域技术人员来说，在不背离本发明精神的范围内所进行的任何显而易见的变化和改进，都应被视为本发明的一部分。

Claims (10)

  1. 一种引物，包含上游引物和下游引物，其特征在于，所述上游引物在SIN载体的3’LTR中的U3区设计，所述下游引物在SIN载体的5’LTR下游的慢病毒骨架设计。
  2. 根据权利要求1所述的引物，其特征在于，所述引物用于扩增序列如SEQ ID NO.1所示的片段、或包括序列与SEQ ID NO.1所示序列互补的序列片段、或包括序列与SEQ ID NO.1所示序列或其互补序列至少一个碱基插入、缺失或替换的序列。
  3. 根据权利要求1所述的检测试剂，其特征在于，所述引物的序列为：
    上游引物U3-F：AGGGCTAATTCACTCCCAACG(SEQ ID NO.2)；
    下游引物LV-R：CGCTTTCAAGTCCCTGTTCG(SEQ ID NO.3)。
  4. 权利要求1～3任一项所述引物在慢病毒滴度检测中的应用。
  5. 一种试剂盒，其特征在于，所述试剂盒包含权利要求1～3任一项所述的引物。
  6. 根据权利要求5所述试剂盒，其特征在于，还包含内参引物。
  7. 根据权利要求6所述试剂盒，其特征在于，所述内参引物的序列为：
    BMP2-F：TAGGGTAGACAGAGCCAAGG(SEQ ID NO.4)；
    BMP2-R：AGCACAGGACAAGAAAGTCATTG(SEQ ID NO.5)。
  8. 根据权利要求5所述的试剂盒，其特征在于，所述检测试剂包含待测样品的标准品。
  9. 根据权利要求5所述的试剂盒，其特征在于，所述检测试剂包含内参片段的标准品。
  10. 一种慢病毒滴度检测方法，其特征在于，包括如下步骤：
    S1：细胞计数并将慢病毒转导至细胞；
    S2：提取细胞DNA；
    S3：用权利要求1～3任一项所述引物或权利要求5～9任一项所述试剂盒进行qPCR扩增，进而分析得到病毒滴度。
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