【作者】 吴蔚；

【导师】 尚世强；

【作者基本信息】 浙江大学 ， 儿科学， 2011， 博士

【摘要】 目的：人巨细胞病毒(Human Cytomegalovirus, HCMV)是人类最常见的先天性感染病毒,在人群中感染率很高,一旦感染后,常不能被机体完全清除,可长期甚至终身携带。目前针对HCMV还没有安全有效的疫苗,而抗HCMV的药物种类有限,目前包括更昔洛韦、西多福韦、膦甲酸、福米韦生、马立巴韦等。但这些药物都是针对病毒侵入靶细胞后的复制翻译阶段起作用,并且有多种毒副作用及耐药性的产生。目前尚缺乏阻遏HCMV侵入靶细胞,即在HCMV与靶细胞膜黏附、结合或者融合等环节起作用的药物研究。甘露糖结合凝集素(mannose-binding lectin, MBL)是一种由肝脏合成的c型凝集素。MBL通过结合病原微生物表面的甘露糖等糖基受体而直接介导调理吞噬作用和/或通过凝集素途径激活补体,在机体的天然免疫防御中发挥重要作用。已有实验室研究发现,MBL对HIV、流感病毒、SARS冠状病毒等都有阻滞作用。同时,临床上也证明,MBL缺陷个体对HIV和HCMV的易感性增加,疾病进展过程加快,继发隐孢子虫病等二重感染率也显著增加。但目前尚无将MBL用于防治HCMV感染的研究。因此,本研究首先构建MBL的真核表达载体,并在哺乳细胞CHO中稳定持续表达,纯化得到重组MBL (rMBL)。然后在体外建立HCMV感染的细胞模型,用不同浓度的rMBL或天然MBL (hMBL)通过不同途径与之相互作用,然后用多种手段综合评价感染情况的变化,从而探讨MBL的抗HCMV作用途径和机制,为HCMV的防治提供新的思路。方法：1. MBL-CDNA TA克隆的建立将MBL序列(747bp) oligo化学合成,将合成好的序列克隆入pCR2.1-TOPO载体并转化至感受态细胞DH5α,挑出阳性重组子菌落小量扩增,快速抽提质粒测序验证重组克隆,确定其包含MBL基因序列。至此得到MBL-CDNA TA克隆。2.真核表达载体PIRES2-AcGFP-MBL的构建以上述来自MBL-CDNA克隆的质粒为模板,进行PCR扩增。扩增引物经设计带有Xhol和EcoRI限制酶切位点。扩增产物经回收、纯化后和目的载体PIRES2-AcGFP分别用Xhol和EcoRI双酶切后连接,连接产物转化感受态细胞DH5a,扩增提取质粒后经双向测序鉴定为目的基因。将构建成功的表达质粒以及空载体质粒分别摇菌扩增,转染级抽提试剂盒抽提质粒,用紫外分光光度计对质粒浓度进行检测。3.CHO细胞的转染和筛选转染：用lipofectamine 2000试剂盒通过脂质体转染法将构建成功的真核表达载体PIRES2-AcGFP-MBL转入CHO-K1细胞,通过观察荧光表达评价转染情况。加药筛选：预先测定CHO细胞对G418的死亡曲线,确定稳转筛选使用G418浓度。将转染后48小时的CHO进行持续加药筛选,每天观察细胞状况以及荧光情况,最后得到稳定转染的细胞株。4. RT-PCR分析稳定转染细胞株MBL基因的表达收集稳转PIRES2-AcGFP-MBL的细胞(CHO-MBL)和空载体的CHO细胞,提取细胞总RNA。采用逆转录试剂盒得到细胞的CDNA,然后行realtime-PCR扩增MBL基因,比较两种细胞内基因的表达量。5.亲和层析法分离纯化MBL将CHO-MBL细胞培养上清先进行离心超滤浓缩,然后进行预处理。将预处理后的样品通过甘露聚糖-Sepharose 4B层析柱进行分离纯化,纯化得到的目的蛋白进行去离子透析后近一步离心超滤浓缩,然后行SDS-PAGE蛋白电泳,考马斯亮蓝染色测定蛋白纯度。天然MBL来自人血浆,纯化步骤同上。6.ELISA法测定重组蛋白浓度采用human mbl ELISA kit,在酶联板中分别加入倍比稀释的rMBL/hMBL样品和不同浓度的标准品,复孔加样。按试剂盒说明反应完成后将酶标板置于自动酶联仪上测定D450nm值,根据标准曲线计算各样本的MBL浓度。7.研究MBL阻滞HCMV入侵细胞的功能HCMV用不同浓度的rMBL/hMBL或加甘露聚糖孵育HCMV,将孵育后的HCMV感染MRC-5细胞2小时,充分洗去未入侵的HCMV,收集细胞,分别用q-PCR和流式细胞术检测细胞内的HCMV-DNA拷贝数和HCMV-PP65阳性率。一部分实验中,将HCMV用PKH-26染色后,再以rMBL/hMBL孵育,然后感染MRC-5细胞,感染后将细胞固定,加入波形蛋白抗体作免疫荧光染色,然后激光共聚焦显微镜下观察感染情况。8.研究MBL阻滞HCMV在细胞间扩散的功能HCMV感染后的MRC-5细胞用不同浓度的rMBL/hMBL或加甘露聚糖共培养3天,每24小时取上清q-PCR检测HCMV-DNA拷贝浓度,第72小时光镜下观察细胞病变效应,然后收集细胞,分别用q-PCR和流式细胞术检测细胞内的HCMV-DNA拷贝数和HCMV-PP65阳性率。结果：1.MBL序列经化学合成后分别克隆入pCR2.1-TOPO载体并转化至感受态细胞DH5a后,快速抽提质粒分别测序验证重组克隆,测序结果与MBL基因序列完全一致,表明MBL-CDNA TA克隆建立成功。2.从已建立MBL-CDNA克隆中提取质粒,通过PCR引入酶切位点,和目的载体PIRES2-AcGFP同样经双酶切后连接,连接产物转化感受态细胞,扩增提取质粒后酶切得到5.3kb(载体),774bp(目的基因)2个片断,目的基因片断经双向测序鉴定包含MBL基因。3.构建成功的表达载体和空载体同时转染CHO细胞,然后按照测定的CHO细胞对G418的死亡曲线,以400μg/ml的G418浓度进行持续加压筛选,3周后,获得2株分别稳定转染空载体和PIRES2-AcGFP-MBL的细胞株,后者命名为CHO-MBL。4.从2种细胞株分别取细胞抽提RNA后进行逆转录,得到CDNA,然后行real-time PCR定量MBL基因的表达,CHO-MBL目的基因表达是转染空载体细胞株的103倍以上。5. CHO-MBL的上清2000ml超滤浓缩成200ml,行亲和层析,收集含蛋白吸脱液约15ml,经PBS透析3天后,再超滤浓缩,得到rMBL1.5ml.然后ELISA检测到1.5ml样品中活性MBL蛋白质浓度为800μg/ml,即原始培养上清中活性rMBL浓度为600μg/L. rMBL进行SDS-PAGE还原电泳,可见32KD单体,非还原电泳均为>170KD之多聚体。6.用梯度浓度的rMBL/hMBL或加甘露聚糖孵育的HCMV感染MRC-5细胞2小时,结果q-PCR检测经10μg/ml rMBL/hMBL孵育的HCMV,侵入细胞的量显著低于未经处理的HCMV,而1μg/ml和5μg/ml rMBL/hMBL孵育的HCMV,侵入细胞的量虽低于未经处理的HCMV,但未达到统计学意义。而经过10μg/ml的rMBL/hMBL和20mg/ml的甘露聚糖同时孵育的HCMV,侵入细胞的量和对照组相比也无显著性差异。同时发现,10μg/ml的hMBL处理组的HCMV感染率明显低于10μg/ml的rMBL处理组的HCMV感染率。激光共聚焦显示感染2小时后病毒颗粒基本位于胞质内,并进一步证实,经10μg/ml的rMBL/hMBL孵育后的病毒,进入细胞的数量明显减少。7. HCMV感染MRC-5细胞后,再和MBL共培养,结果经q-PCR检测,经10μg/ml的rMBL/hMBL孵育的HCMV,在培养第24小时和第48小时,各实验组培养上清内HCMV-DNA拷贝数低于对照组,但未达到统计学强度。而在培养第72小时细胞内的量和培养上清中的量均显著低于未经处理的HCMV,而1μg/ml和5μg/ml的rMBL/hMBL孵育的HCMV和未经处理的HCMV相比,结果无显著性差异。同时比较细胞内PP65阳性率以及细胞病变效应,10μg/ml的rMBL/hMBL孵育组也低于对照组。结论：1.获得了表达人MBL的CHO细胞株和有较强生物活性的rMBL。2. rMBL在CHO细胞中的产量为600μg/L,多为>170KD的多聚体,有较强生物活性。3.MBL在细胞外液中的浓度达到10μg/ml时候能阻滞HCMV入侵细胞,作用达到一定时间后能够阻滞已入侵HCMV在细胞间传播和增殖。4.MBL可能通过糖结合区和病毒的糖蛋白结合,阻遏病毒吸附入侵细胞而起抗病毒作用。更多还原

【Abstract】 Objective:Human cytomegalovirus (HCMV) is the leading viral cause of congenital disorders and has high sero-prevalence rates among human. Primary HCMV infection occurs early in life and followed by the establishment of a life long latency in the infected host. A primary infection or reactivation of endogenous virus in immunosuppressive individuals is associated with substantial morbidity and mortality. To date, a limited number of drugs have been used for treatment of HCMV and disease, including ganciclovir, cidofovir, foscamet, fomiversen and maribavir. These drugs all share the similar antiviral mechanism. Although combating HCMV by inhibiting virus replication is effective and has been established for many years, problems with toxicity and emergence of drug resistance underscore the need to develop new and improved antiviral drugs with novel molecular targets. The blocking of virus entry into cells before their replication is a promising way to research new antiviral agents.Mannose-binding lectin (MBL), a soluble C-type lectin, constitutes an important part of the innate immune defence. MBL binds to repetitive mannose and N-acetylglucosamine residues on microorganisms, activating the complement system. MBL can inhibit the infection of HIV, influenza A virus and SARS-CoV. MBL deficiency may lead to susceptibility to HCMV and secondary infection. However, the antiviral activities of human MBL against HCMV are not well studied.This study expressed human MBL gene in mammary cells and obtained recombinant MBL (rMBL). Then the anti-HCMV activities of both rMBL and isolated native MBL (hMBL) from human serum were tested and the possible mechanisms were explored.Methods:1. Establishment of MBL-CDNA TA cloneThe target sequence of MBL (747bp) was produced by oligo synthesis, and the gene was cloned to pCR2.1-TOPO vector and then transformed to competent cell DH5a. The transformed cells were proliferated and the plasmids were extracted and sequenced to confirm the MBL-CDNA clone was established.2. Construction of recombinant expression vector PIRES2-AcGFP-MBLThe above plasmids from MBL-CDNA clone were amplified and the restriction sites for XhoI and EcoRI were added by PCR. The PCR products and the expression vector PIRES2-AcGFP were both cleaved by endonucleases XhoI and EcoRI and followed by the sequential insertions of the MBL genes into the expression vector PIRES2-AcGFP. After transformation to E. coli DH5a, the recombinants were selected, amplified and sequenced. The interested sequences were then compared with the MBL gene sequences to confirm the correct insertion.3. Stable transfection of CHO cells by recombinant expression vector PIRES2-AcGFP-MBLThe recombinant expression vector PIRES2-AcGFP-MBL and the empty vector were transfected to CHO cells by using lipofectamine 2000 kit. After a sensitive toxicity test, G418 with certain dilution were put into cell culture medium and cell morphological changes and green fluorescence were observed every day until G418-resistant cell clones were selected. These clones were stable transfected by recombinant expression vector.4. RT-PCR analyse MBL expression in stable transfedted CHO cellsTotal cell RNAs were extracted and were reverse transcripted to CDNA. The MBL expression level in PIRES2-AcGFP-MBL transfected CHO cells were compared t to that in empty vector transfected CHO cells.5. Purificatioin of MBL protein by affinity chromatographyThe rMBL were purified utilizing the calcium-dependent carbohydrate binding by MBL. The culture supernatant of stable transformants was concentrated by centrifugal ultrafiltration and pretreated before pass a chromatography column packed with mannose-Sepharose. The bound MBL were eluted and concentrated and then identified by SDS-PAGE electrophoresis and Commassie Blue Staining.6. Quantitation of MBL by ELISAThe ELISA procedure was performed according to the Hbt human MBL ELISA kit and the absorbance was measured and the MBL level of tested sample was calculated according to standard curve.7. HCMV neutralization testHCMV was treated with each diluted MBL solution or both with mannan, and inoculated onto the MRC-5 cell monolayers for 2 hours. The cells were then washed and the HCMV-DNAs in cells were quantified by Q-PCR and the HCMV-PP65 antigens were tested by flow cytometry (FCM). Cells infected by PKH-26 stained HCMV were fixed and anti-vimentin was used to obtain immunofluorescence. The cells and HCMV were observed by confocal microscope.8. HCMV growth inhibition testAfter HCMV inoculation onto monolayers of MRC-5 cell for 2 hours, the cells were washed and incubated with several dilutions of MBL or with mannan at the same time. Then the cells were incubated for 3 days, every 24 hours, the supernatant of cell culture was tested for HCMV-DNA by Q-PCR. At 72 hour, cytopathic effect (CPE) were observed by the inverted microscope and cells were collected for HCMV-DNA quantitation and HCMV-PP65 test.Results:1. The sequence of extracted plasmid from pCR2.1-TOPO vector transformed cell DH5αclone were consistent with the MBL sequence and confirmed the establishment of MBL-CDNA TA clone.2. The MBL sequence from MBL-CDNA clone was inserted into expression vector. The resulting recombinant vector was transformed to DH5αcell to proliferation and then extracted and digested with endonuclease and two correct sequences were obtained with one is 5.3kb (vector) and one is 774bp (target gene).3. The recombinant expression vector and empty vector was transinfected into CHO cells.400μg/ml G418 was used to select resistant cells according to sensitive toxicity test. After 3 weeks, two cell lines transfected with recombinant expression vector and empty vector separately were obtained. The former was named as CHO-MBL.4. The total RNAs extracted from the above 2 cell lines were reverse transcripted and MBL gene were amplified by Q-PCR. The MBL expression level in CHO-MBL was 103 times higher than the empty vector transfected cell.5. CHO-MBL culture supernatant was concentrated from 2000ml to 200ml and 1.5ml rMBL obtained after affinity chromatography and enrich. The MBL concentration of the above 1.5ml sample was 800μg/ml according to ELISA. So the MBL concentration in culture supernatant was 600μg/L. SDS-PAGE electrophoresis and followed Commassie Blue Staining showed rMBL was a 32-KD protein under reducing condition and multimer>170KD under unreducing condition.6. HCMV neutralization test revealed 10μg/ml rMBL/hMBL significantly decreased the HCMV invasion in MRC-5 cell. However, the invasion of HCMV incubated with both 10μg/ml rMBL/hMBL and 20mg/ml mannan had no difference with untreated HCMV. 1μg/ml and 5μg/ml rMBL/hMBL have no such function. The infective rate of 10u.g/ml rMBL incubated HCMV was higher than 10μg/ml hMBL incubated HCMV. Confocal pictures showed after 2 hours of HCMV invasion, the virus were mostly located in cytoplasma and less HCMV invasion after MBL treatment were confirmed.7. HCMV growth inhibition test showed at 24th hour and 48th hour after HCMV invasion, there was no difference in the HCMV-DNA concentrations between MBL incubated cells supernatant and control cells supernatant. At 72nd hour, the HCMV-DNA concentrations in supernatant of 10μg/ml rMBL/hMBL incubated cells were lower than control cells. After 3 days of infection, both HCMV-DNA and HCMV-PP65 in 10μg/ml rMBL/hMBL incubated cells were lower than control cells.Conclusin:1. The CHO cell line express rMBL and the purified rMBL with high bioactivity were obtained.2. The concentration of rMBL in cell supernatant was 600μg/L and the majority of the purified rMBL was estimated to contain multimers larger than 170 KD.3.10μg/ml MBL can inhibit invasion of HCMV and prevents viral spreading to contiguous cells after 72 hours.4. MBL inhibit HCMV infection by binding the glucoprotein of HCMV and blocking its attachement and invasion to cytomembrane.更多还原