【作者】 孙照刚；

【导师】 张曼夫；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 以纯化的传染性喉气管炎病毒(ILTV)北京E3株基因组为模板,通过PCR方法扩增了E3毒株的完整的糖蛋白G(gG)基因及其两侧的序列,包括部分UL47同源序列、完整的US4同源序列(gG基因)以及部分的ORF5序列,将其克隆到pGEM-T载体上,构建了pGEM-FgG质粒,并进行了测序。通过设计另外一对引物克隆了Zhonghai弱毒疫苗株的gG基因,并进行了测序,发现E3株与Zhonghai株的gG基因序列完全一致。与其它疱疹病毒的gG基因和推测的蛋白序列进行了的进化关系、蛋白同源序列比较发现gG蛋白在ILTV中高度保守,疱疹病毒gG蛋白都存在三个保守的半胱氨酸位点,并且三个位点之间间隔的氨基酸残基相同。等电点以及天然蛋白可能存在的修饰等方面的分析,为进一步研究该蛋白的功能奠定了基础。 利用两种大肠杆菌表达系统(pMAL-c2x(E. coli BT1)和pET-30a(+)(E. coli BL21)载体系统),对不含信号肽部分的gG基因进行了原核表达,结果表明这两种表达系统都具有较高的表达水平,pMAL-c2x(E. coli BT1)表达系统中目的蛋白(MBP-GG)占整个菌体蛋白的比例为25.8%,但是gG蛋白占整个MBP-GG融合蛋白的比例不超过45%;pET-30a(+)(E. coliBL21)表达系统中目的蛋白(His-GG)占整个菌体蛋白的比例为18.3%,组氨酸标签占His-GG融合蛋白的比例极小,约10%左右。其中MBP-GG融合蛋白是可溶性表达的,分子量大约为72KDa;His-GG融合蛋白是以包涵体的形式存在的,需要使用脲进行变性和复性,分子量为34KDa。经分离纯化分别得到两种融合蛋白MBP-GG和His-GG后,分别制备了抗ILTV gG蛋白的大鼠抗血清,并利用蛋白G对两种抗血清进行了纯化,获得了纯化的免疫球蛋白。 为了进一步研究gG蛋白的功能,通过利用DEAE BIO-GEL从感染ILTV的鸡胚尿囊液中纯化得到ILTV天然gG蛋白。利用在鸡胚原代单层肝细胞(CEL)培养液中添加ILTV天然gG蛋白及其抗体的方法,对gG蛋白在ILTV(北京E3和Zhonghai)的吸附、侵入、细胞到细胞间的直接传染以及病毒的复制曲线方面的作用进行了研究,结果表明:gG蛋白在病毒的吸附和侵入过程中没有作用,但是北京E3和Zhonghai两毒株都具有较强的吸附和侵入效率,两毒株的吸附效率分别为对照的85.6%和79.1%;两毒株的侵入效率分别为68.9%和62.9%。添加抗体能够降低在细胞上培养的病毒滴度,并促使ILTV在CEL单层细胞上形成的噬斑明显变小,分别为对照的50.96%(E3)和56.73%(Zhonghai)。gG蛋白的添加在上述几个方面都没有发现可见的作用,推测可能的原因是ILTVgG蛋白不能单独起作用,需要有其它蛋白分子协同作用;也可能是由于病毒gG蛋白在CEL细胞中的表达特点所致,gG基因启动子属于中早期启动子,并且启动效率高,病毒活动产生的gG蛋白掩盖了添加部分的gG蛋白的功能。 利用前面克隆的完整的gG基因及其两侧的序列,将绿色荧光蛋白(GFP)基因插入gG基因ORF中间,分别构建了以CMV、SV40和ILTV gG基因启动子作为启动子,以SV40 polyA作为终止序列,用以启动GFP基因表达的转移质粒pGEM-FgG/CMV-GFP、pGEM-FgG/SV40-GFP和pGEM-FgG/GFP。利用这三种转移质粒和pBL-ICP4质粒与ILTV基因组DNA共转染原代CEL细胞,24h后,用荧光显微镜检测带荧光的ILTV重组子,结果发现:更多还原

【Abstract】 The glycoprotein G （gG） gene and its flank sequences, including part of HSV-I UL47 homologue, complete gG gene and ORF5, of infectious laryngotracheitis virus （ILTV） of virulent Beijing E3 strain was amplified by PCR, cloned into pGEM-T vector and sequenced. The gG gene of ILTV vaccine strain Zhonghai was also amplified with another pair of primers and sequenced. Sequence analyses showed that the gG gene was very closed among sequenced ILTV strains. The deduced amino acids of most alphaherpesviruses’ gG proteins were very conservative. They have a characteristic pattern of three closely-spaced cysteine residues. Other characteristics of gG protein were also analysed such as hydrophilicity, antigenic index, titration curve and the modification by phosphorylation sulfation glycosylation.The pMAL-c2x （E.coli BT1） and pET-30a（+） （E.coli BL21） prokaryotic expression system were used to express the gG excluding the signal sequence. The results showed that two systems showed high expression level. The pMAL-c2x （E.coli BT1） system expressed the soluble fusion protein MBP-GG was 25.8 percent of total bacterial protein and the Mr was about 72KDa of which the GG takes less 45 percent. The pET-30a（+） （E.coli BL21） system expressed the insoluble fusion protein His-GG was 18.3 of total bacterial protein and the Mr was about 34KDa of which the GG takes about 90 percent. After purification, those two kinds of fusion proteins were immunized the rats respectively to prepare their antibodies. Then the antibody was purified.The gG protein of the ILTV was purified from the allantoid fluid of the chicken’s embryo by using the DEAE BIO-GEL. By adding the gG and its antibody to the culture medium of the primary CEL cell monolayers, the ILTV gG function was analyzed in ILTV attachment, penetration, direct cell-to-cell spread （CTCS） and the growth curve. The treatment with ILTV gG had no obvious affection to the virus infection, penetration, CTCS and growth curve. The attachment efficiencies of the E3 and Zhonghai were 85.6 and 79.1 percent respectively, and the penetration efficiencies of the E3 and Zhonghai were 68.9 and 62.9 percent respectively. The E3 and Zhonghai strains of the ILTV showed the characters of strong attachment and penetration. The gG antibody reduced the virus plaque size （50.96 percent （E3） and 56.73 percent （Zhonghai） of the control） and the one-step growth curve on CEL cells, but the gG treatment did not. There were several reasons for this result. One was that the ILTV gG was expressed at early and middle phase （data not shown） and was expressed strongly, which attenuated the function of the additive gG Little effect of the gG addition to the culture medium may also imply that the gG couldn’t work by itself. Laser scanning confocal microscopy （LSCM） detection showed that the ILTV gG lied in perinuclear region and membrane of the CEK cells. Those results suggested that the ILTV gG might work in direct cell-to-cell transmission.The plasmids of pGEM-FgG/CMV-GFP, pGEM-FgG/SV40-GFP and pGEM-FgG/GFP expressing the green fluorescent protein （GFP） were constructed with the CMV、 SV40 and the ILTV gG promoter as the promoter respectively and the SV40 polyA as the stop codes. By the homologous recombination method the expressing frames was inserted into the ILTV genome to replace the gG gene respectively. The recombinants of ILTV were detected by using fluorescent microscope. The stability of recombinants of ILTV was detected by a serial passage on chicken liver cells. The results showed that the plasmids, pGEM-FgG/CMV-GFP, pGEM-FgG/SV40-GFP and pGEM-FgG/GFP, could express the GFP and that the recombinant alive ILTV could be observed after three generations, the more generation we passed the less recombinant ILT virions that were observed and that the gG promoter need the ILTV protein（s） to express the gene. Those results suggested that the cotransfection of the lined plasmids pBL-ICP4, pGEM-FgG/CMV-GFP, pGEM-FgG/SV40-GFP and pGEM-FgG/GFP and the ILTV DNA genome could generate the recombinant alive ILT virions (ILTV ^-gG), but更多还原