HPV-16在HEK293T細(xì)胞中的復(fù)制以及HPV-16 L1病毒載體疫苗免疫效果的研究
發(fā)布時(shí)間：2018-07-15 17:45
【摘要】：人乳頭狀瘤病毒(human papillomavirus,HPV)為無(wú)包膜的小型雙鏈環(huán)狀DNA病毒,可引起皮膚和粘膜的良性和惡性腫瘤,基因組全長(zhǎng)約8kb,分為早期區(qū)(E區(qū))、晚期區(qū)(L區(qū))和上游調(diào)控區(qū)(Upstream Regulatory Region,URR)。HPV以人為單一宿主,具有明顯的種屬特異性,且HPV病毒體僅存在于終末分化的上皮組織中,含量很少,很難在體外培養(yǎng)的細(xì)胞中增殖,這也阻礙了HPV基礎(chǔ)研究和疫苗研制。本研究以新疆株HPV-16全長(zhǎng)基因組的質(zhì)粒pTXJHPV-16轉(zhuǎn)染HEK293T細(xì)胞,以探討HPV-16在體外培養(yǎng)細(xì)胞中的復(fù)制。 pTXJHPV-16單獨(dú)或與pcDNA3.1-L1L2共同轉(zhuǎn)染HEK293T細(xì)胞,以及線性化的pTXJHPV-16單獨(dú)或與pcDNA3.1-L1L2共同轉(zhuǎn)染HEK293T細(xì)胞,均能產(chǎn)生病毒樣顆粒,且pTXJHPV-16單獨(dú)轉(zhuǎn)染細(xì)胞產(chǎn)生的病毒顆粒較多。繼而針對(duì)pTXJHPV-16單獨(dú)轉(zhuǎn)染HEK293T細(xì)胞以研究HPV-16的復(fù)制,PCR檢測(cè)HPV-16在HEK293T細(xì)胞中的存在狀態(tài);Southern blotting檢測(cè)轉(zhuǎn)染細(xì)胞2 h、20 h、40 h、60 h后,HPV-16基因組在HEK293T細(xì)胞中的復(fù)制及穩(wěn)定存在的時(shí)間;DpnⅠ酶切pTXJHPV-16轉(zhuǎn)染2 h、20 h、40 h的細(xì)胞基因組,Southern blotting檢測(cè)新合成的病毒DNA;RT-PCR檢測(cè)轉(zhuǎn)染細(xì)胞中HPV-16 E1、L1和E1^E4轉(zhuǎn)錄體。 結(jié)果表明,轉(zhuǎn)染細(xì)胞中存在完整的E1、E2基因,推測(cè)HPV-16以游離的形式存在。Southern blotting檢測(cè)發(fā)現(xiàn)HPV-16基因組在轉(zhuǎn)染20 h時(shí)有所減少,并持續(xù)到40 h,推測(cè)是細(xì)胞內(nèi)HPV-16基因組被DNA酶消化;而在60 h后HPV-16基因組的量開(kāi)始增加,說(shuō)明HPV-16基因組在轉(zhuǎn)染細(xì)胞中得以復(fù)制。Southern blotting檢測(cè)表明轉(zhuǎn)染20 h后存在新合成的未被甲基化的HPV-16基因組,進(jìn)一步說(shuō)明HPV-16基因組在HEK293T細(xì)胞中可短暫復(fù)制。RT-PCR檢測(cè)到E1、L1、E1^E4轉(zhuǎn)錄體,表明HPV-16基因能在HEK293T細(xì)胞中表達(dá)。以上結(jié)果表明HPV-16能夠在HEK293T細(xì)胞中復(fù)制。 HPV主要衣殼蛋白L1具有較強(qiáng)的免疫原性,能夠自組裝形成病毒樣顆粒,是宮頸癌預(yù)防性疫苗的理想靶抗原。l型單純皰疹病毒(Herpes simplex virus type 1,HSV-1)為有包膜的DNA病毒,其作為載體具有基因容量大、宿主范圍廣、可高效率地感染分裂和非分裂細(xì)胞等優(yōu)勢(shì)。本研究利用HSV-1載體高效表達(dá)新疆株HPV-16 L1基因,以獲得HPV-16 L1病毒載體疫苗。 前期研究已獲得了刪除HSV-1內(nèi)部反向重復(fù)序列的重組病毒HSV-1/BN。本研究將HPV-16 L1基因插入質(zhì)粒pKO5/BN,獲得重組質(zhì)粒pKO5/BN/L1,并電轉(zhuǎn)至含有BAC-HSV的大腸桿菌細(xì)胞中,經(jīng)過(guò)溫度和抗生素篩選獲得重組DNA BAC-HSV-L1,將其轉(zhuǎn)染至Vero細(xì)胞中,篩選并純化獲得重組病毒HSV-1/L1,測(cè)定病毒滴度。分別通過(guò)滴眼、肌肉和皮下接種BALB/c小鼠,接種病毒量依次為1×105、1×10~6和1×10~6 pfu,連續(xù)免疫三次,每次間隔14d,同時(shí)設(shè)野生型HSV-1免疫對(duì)照組。分別于每次免疫前和第一次免疫后42d采小鼠血清, ELISA檢測(cè)L1特異性的IgG水平。PCR、Southern blotting檢測(cè)表明重組DNA BAC-HSV-L1構(gòu)建正確,其在轉(zhuǎn)染細(xì)胞后獲得的重組病毒HSV-1/L1能在Vero細(xì)胞中大量增殖,測(cè)定其滴度為2.2×107;HSV-1/L1滴眼免疫組血清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組血清中L1抗體水平均極顯著高于HSV-1/WT免疫組,說(shuō)明HSV-1/L1通過(guò)眼瞼免疫能夠激發(fā)體液免疫反應(yīng)。肌肉和皮下接種均沒(méi)有激發(fā)體液免疫反應(yīng)。以上結(jié)果為HSV-1載體疫苗的研究奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：新疆大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：新疆大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 馬正海,張富春,梅新娣,馬彩玲,劉開(kāi)江;新疆南部地區(qū)維吾爾族婦女宮頸癌組織中人乳頭狀瘤病毒16型L1基因突變譜分析[J];中華醫(yī)學(xué)雜志;2004年12期
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