HPV-16在HEK293T细胞中的复制以及HPV-16 L1病毒载体疫苗免疫效果的研究
发布时间：2018-07-15 17:45
【摘要】：人乳头状瘤病毒(human papillomavirus,HPV)为无包膜的小型双链环状DNA病毒,可引起皮肤和粘膜的良性和恶性肿瘤,基因组全长约8kb,分为早期区(E区)、晚期区(L区)和上游调控区(Upstream Regulatory Region,URR)。HPV以人为单一宿主,具有明显的种属特异性,且HPV病毒体仅存在于终末分化的上皮组织中,含量很少,很难在体外培养的细胞中增殖,这也阻碍了HPV基础研究和疫苗研制。本研究以新疆株HPV-16全长基因组的质粒pTXJHPV-16转染HEK293T细胞,以探讨HPV-16在体外培养细胞中的复制。 pTXJHPV-16单独或与pcDNA3.1-L1L2共同转染HEK293T细胞,以及线性化的pTXJHPV-16单独或与pcDNA3.1-L1L2共同转染HEK293T细胞,均能产生病毒样颗粒,且pTXJHPV-16单独转染细胞产生的病毒颗粒较多。继而针对pTXJHPV-16单独转染HEK293T细胞以研究HPV-16的复制,PCR检测HPV-16在HEK293T细胞中的存在状态;Southern blotting检测转染细胞2 h、20 h、40 h、60 h后,HPV-16基因组在HEK293T细胞中的复制及稳定存在的时间;DpnⅠ酶切pTXJHPV-16转染2 h、20 h、40 h的细胞基因组,Southern blotting检测新合成的病毒DNA;RT-PCR检测转染细胞中HPV-16 E1、L1和E1^E4转录体。 结果表明,转染细胞中存在完整的E1、E2基因,推测HPV-16以游离的形式存在。Southern blotting检测发现HPV-16基因组在转染20 h时有所减少,并持续到40 h,推测是细胞内HPV-16基因组被DNA酶消化;而在60 h后HPV-16基因组的量开始增加,说明HPV-16基因组在转染细胞中得以复制。Southern blotting检测表明转染20 h后存在新合成的未被甲基化的HPV-16基因组,进一步说明HPV-16基因组在HEK293T细胞中可短暂复制。RT-PCR检测到E1、L1、E1^E4转录体,表明HPV-16基因能在HEK293T细胞中表达。以上结果表明HPV-16能够在HEK293T细胞中复制。 HPV主要衣壳蛋白L1具有较强的免疫原性,能够自组装形成病毒样颗粒,是宫颈癌预防性疫苗的理想靶抗原。l型单纯疱疹病毒(Herpes simplex virus type 1,HSV-1)为有包膜的DNA病毒,其作为载体具有基因容量大、宿主范围广、可高效率地感染分裂和非分裂细胞等优势。本研究利用HSV-1载体高效表达新疆株HPV-16 L1基因,以获得HPV-16 L1病毒载体疫苗。 前期研究已获得了删除HSV-1内部反向重复序列的重组病毒HSV-1/BN。本研究将HPV-16 L1基因插入质粒pKO5/BN,获得重组质粒pKO5/BN/L1,并电转至含有BAC-HSV的大肠杆菌细胞中,经过温度和抗生素筛选获得重组DNA BAC-HSV-L1,将其转染至Vero细胞中,筛选并纯化获得重组病毒HSV-1/L1,测定病毒滴度。分别通过滴眼、肌肉和皮下接种BALB/c小鼠,接种病毒量依次为1×105、1×10~6和1×10~6 pfu,连续免疫三次,每次间隔14d,同时设野生型HSV-1免疫对照组。分别于每次免疫前和第一次免疫后42d采小鼠血清, ELISA检测L1特异性的IgG水平。PCR、Southern blotting检测表明重组DNA BAC-HSV-L1构建正确,其在转染细胞后获得的重组病毒HSV-1/L1能在Vero细胞中大量增殖,测定其滴度为2.2×107;HSV-1/L1滴眼免疫组血清ELISA分析表明,免疫后14 d(P 0.01)、28 d(P 0.001)、42 d(P 0.001) HSV-1/L1组血清中L1抗体水平均极显著高于HSV-1/WT免疫组,说明HSV-1/L1通过眼睑免疫能够激发体液免疫反应。肌肉和皮下接种均没有激发体液免疫反应。以上结果为HSV-1载体疫苗的研究奠定了基础。
[Abstract]:Human papillomavirus (human papillomavirus, HPV) is a small double stranded circular DNA virus without membrane. It can cause benign and malignant tumors of the skin and mucous membrane. The total length of the genome is about 8KB, which is divided into early region (E region). The late region (L region) and the upstream regulatory region (Upstream Regulatory Region, URR).HPV are a single host and have obvious species. Heterosexual, and HPV virus body only exists in terminal differentiated epithelial tissue, and it is very difficult to proliferate in the cells cultured in vitro. This also hinders the basic research and vaccine development of HPV. This study transfected HEK293T cells with the plasmid pTXJHPV-16 of the full-length genome of Xinjiang strain HPV-16 to explore the replication of HPV-16 in the cultured cells.
PTXJHPV-16 alone or pcDNA3.1-L1L2 co transfected HEK293T cells, as well as linear pTXJHPV-16 alone or co transfected with pcDNA3.1-L1L2 cells, can produce virus like particles, and pTXJHPV-16 alone transfected cells produced more virus particles. Then pTXJHPV-16 alone transfected HEK293T cells to study the replication of HPV-16. PCR detected the existence state of HPV-16 in HEK293T cells; Southern blotting detected the replication and stable existence of HPV-16 genome in HEK293T cells after transfection of 2 h, 20 h, 40 h, and 60 h; Dpn I enzyme was transfected to 2, 20, 40 cells. HPV-16 E1, L1 and E1^E4 transcripts in the cells.
The results showed that there was a complete E1, E2 gene in the transfected cells, and that the.Southern blotting detection in the free form of HPV-16 found that the HPV-16 genome was reduced when transfected to 20 h and continued to 40 h. It was presumed that the HPV-16 genome in the cells was digested by DNA enzyme, and the amount of the HPV-16 genome began to increase after 60 h, indicating the genome. The replication of.Southern blotting in transfected cells showed the existence of a newly synthesized non methylation HPV-16 genome after 20 h transfection, and further indicated that the HPV-16 genome could be briefly replicated in HEK293T cells by.RT-PCR to detect E1, L1, E1^E4 transcript, indicating that the HPV-16 gene could be expressed in the HEK293T cells. The above results showed that the HPV-16 genome was expressed in the HEK293T cells. It can be replicated in HEK293T cells.
HPV main capsid protein L1 has strong immunogenicity and can self assemble to form virus like particles. It is an ideal target antigen for the prophylactic vaccine of cervical cancer,.L herpes simplex virus (Herpes simplex virus type 1, HSV-1) as a coated DNA virus. As a carrier, it has a large gene capacity and a wide range of host, which can efficiently infect and divide and split the virus. In this study, the HPV-16 L1 gene of Xinjiang strain was highly expressed by HSV-1 vector, and HPV-16 L1 virus vector vaccine was obtained.
The previous study has obtained the recombinant virus HSV-1/BN. that deletes the internal reverse repeat sequence of the HSV-1. The HPV-16 L1 gene was inserted into the plasmid pKO5/BN, and the recombinant plasmid pKO5/BN/L1 was obtained and transferred to the Escherichia coli cells containing BAC-HSV. The recombinant DNA BAC-HSV-L1 was obtained through the screening of the temperature and antibiotics and transfected into Vero cells. The recombinant virus HSV-1/L1 was selected and purified to determine the titer of the virus. BALB/c mice were inoculated with 1 x 105,1 x 10~6 and 1 x 10~6 PFU respectively by eye drop, muscle and subcutaneous inoculation, respectively, for three consecutive immunizations, 14d at each interval, and the wild type HSV-1 immune control group. The mice were separated from the blood before and after the first immunization. ELISA detected L1 specific IgG level.PCR, and Southern blotting detection showed that recombinant DNA BAC-HSV-L1 was constructed correctly. The recombinant virus HSV-1/L1 could proliferate in Vero cells after transfection of cells, and its titer was 2.2 x 107; HSV-1/L1 eye immunization group showed that 14 (0.01) after immunization, 28 (0.001). The serum levels of L1 antibody in the 42 d (P 0.001) HSV-1/L1 group were significantly higher than those in the HSV-1/WT immunization group, indicating that HSV-1/L1 could stimulate the humoral immune response through the eyelid immunization. Both muscle and subcutaneous inoculation did not stimulate the humoral immune response. The above results lay a foundation for the study of the HSV-1 carrier vaccine.
【学位授予单位】：新疆大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2124871
[Abstract]:Human papillomavirus (human papillomavirus, HPV) is a small double stranded circular DNA virus without membrane. It can cause benign and malignant tumors of the skin and mucous membrane. The total length of the genome is about 8KB, which is divided into early region (E region). The late region (L region) and the upstream regulatory region (Upstream Regulatory Region, URR).HPV are a single host and have obvious species. Heterosexual, and HPV virus body only exists in terminal differentiated epithelial tissue, and it is very difficult to proliferate in the cells cultured in vitro. This also hinders the basic research and vaccine development of HPV. This study transfected HEK293T cells with the plasmid pTXJHPV-16 of the full-length genome of Xinjiang strain HPV-16 to explore the replication of HPV-16 in the cultured cells.
PTXJHPV-16 alone or pcDNA3.1-L1L2 co transfected HEK293T cells, as well as linear pTXJHPV-16 alone or co transfected with pcDNA3.1-L1L2 cells, can produce virus like particles, and pTXJHPV-16 alone transfected cells produced more virus particles. Then pTXJHPV-16 alone transfected HEK293T cells to study the replication of HPV-16. PCR detected the existence state of HPV-16 in HEK293T cells; Southern blotting detected the replication and stable existence of HPV-16 genome in HEK293T cells after transfection of 2 h, 20 h, 40 h, and 60 h; Dpn I enzyme was transfected to 2, 20, 40 cells. HPV-16 E1, L1 and E1^E4 transcripts in the cells.
The results showed that there was a complete E1, E2 gene in the transfected cells, and that the.Southern blotting detection in the free form of HPV-16 found that the HPV-16 genome was reduced when transfected to 20 h and continued to 40 h. It was presumed that the HPV-16 genome in the cells was digested by DNA enzyme, and the amount of the HPV-16 genome began to increase after 60 h, indicating the genome. The replication of.Southern blotting in transfected cells showed the existence of a newly synthesized non methylation HPV-16 genome after 20 h transfection, and further indicated that the HPV-16 genome could be briefly replicated in HEK293T cells by.RT-PCR to detect E1, L1, E1^E4 transcript, indicating that the HPV-16 gene could be expressed in the HEK293T cells. The above results showed that the HPV-16 genome was expressed in the HEK293T cells. It can be replicated in HEK293T cells.
HPV main capsid protein L1 has strong immunogenicity and can self assemble to form virus like particles. It is an ideal target antigen for the prophylactic vaccine of cervical cancer,.L herpes simplex virus (Herpes simplex virus type 1, HSV-1) as a coated DNA virus. As a carrier, it has a large gene capacity and a wide range of host, which can efficiently infect and divide and split the virus. In this study, the HPV-16 L1 gene of Xinjiang strain was highly expressed by HSV-1 vector, and HPV-16 L1 virus vector vaccine was obtained.
The previous study has obtained the recombinant virus HSV-1/BN. that deletes the internal reverse repeat sequence of the HSV-1. The HPV-16 L1 gene was inserted into the plasmid pKO5/BN, and the recombinant plasmid pKO5/BN/L1 was obtained and transferred to the Escherichia coli cells containing BAC-HSV. The recombinant DNA BAC-HSV-L1 was obtained through the screening of the temperature and antibiotics and transfected into Vero cells. The recombinant virus HSV-1/L1 was selected and purified to determine the titer of the virus. BALB/c mice were inoculated with 1 x 105,1 x 10~6 and 1 x 10~6 PFU respectively by eye drop, muscle and subcutaneous inoculation, respectively, for three consecutive immunizations, 14d at each interval, and the wild type HSV-1 immune control group. The mice were separated from the blood before and after the first immunization. ELISA detected L1 specific IgG level.PCR, and Southern blotting detection showed that recombinant DNA BAC-HSV-L1 was constructed correctly. The recombinant virus HSV-1/L1 could proliferate in Vero cells after transfection of cells, and its titer was 2.2 x 107; HSV-1/L1 eye immunization group showed that 14 (0.01) after immunization, 28 (0.001). The serum levels of L1 antibody in the 42 d (P 0.001) HSV-1/L1 group were significantly higher than those in the HSV-1/WT immunization group, indicating that HSV-1/L1 could stimulate the humoral immune response through the eyelid immunization. Both muscle and subcutaneous inoculation did not stimulate the humoral immune response. The above results lay a foundation for the study of the HSV-1 carrier vaccine.
【学位授予单位】：新疆大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
【参考文献】
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1 马正海,张富春,梅新娣,马彩玲,刘开江;新疆南部地区维吾尔族妇女宫颈癌组织中人乳头状瘤病毒16型L1基因突变谱分析[J];中华医学杂志;2004年12期
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