摘要
猪瘟(Classical swine fever, CSF)是由猪瘟病毒(Classical swine fever virus, CSFV)引起猪群爆发的一种重要的烈性传染病,对养猪业危害极大。预防CSFV感染的有效方法是采用疫苗免疫,建立稳定而有效的中和抗体检测方法是评价疫苗免疫效果的重要手段。
猪瘟伪型病毒(VSV△G*CSF),是指用表达绿色荧光蛋白(GFP)的报告基因代替水疱性口炎病毒(Vesicular stomatitis virus,VSV)基因组中编码囊膜蛋白G的基因,从而形成含有报告基因的复制缺陷型病毒(VSV△G*);当外源提供CSFV囊膜蛋白时,可与VSV△G*重新装配,整合成具有一过性感染力的VSV重组病毒VSV△G*CSF。该病毒拥有VSV的基因组,囊膜上整合有CSFV的囊膜蛋白,因此具有了CSFV的感染特征,这种基因型和表现型不一致的现象叫伪型,具有这种特征的病毒叫猪瘟伪型病毒。
本研究通过构建表达CSFV全部囊膜蛋白的重组真核表达质粒pCAGG-E012顺式表达出CSFV囊膜蛋白Erns、E1和E2。结合已有对瘟病毒的报道,通过生物软件进行比对与分析后,推测位于CSFV囊膜蛋白E2基因跨膜区内的膜锚定序列是阻碍其表达在细胞表面的主要因素,对E2锚定序列进行部分缺失后发现:E2锚定序列的部分缺失会减少E2蛋白在细胞内的表达,说明锚定序列对E2的正确表达意义重大。只有表达全部CSFV囊膜蛋白,才能够研制出滴度相对较高的猪瘟伪型病毒VSV△G*CSF,其病毒的滴度为10~2～10~3 pfu·mL~(-1),且该病毒能够被抗CSFV的阳性血清所中和。
传统检测猪瘟病毒中和抗体的ELISA以及间接免疫荧光方法普遍存在费时、繁琐、不够精确、特异性差等一系列缺陷,亟待改进。为了提高CSFV中和抗体检测的效率和生物安全性、研究CSFV的侵染机制,本研究制备出含有报告基因的复制缺陷型VSV/CSFV伪型病毒(VSV△G*CSF)作为代替野生型CSFV的感染模型进行中和试验,使检测过程更加安全、简单,结果评定更加直观、有效。经研究表明,200 pfu的VSV△G*CSFV可被150倍稀释的标准猪瘟阳性血清所中和,利用该方法检测10份现地待检样品,结果显示7份为阳性,3份为阴性,与商品化试剂盒检测结果的符合率为90%,表明该方法能够作为一种新型的检测工具,应用于猪瘟中和抗体检测。同时,本研究对传统利用重组VSV系统制备伪型病毒的方法进行改良,节省了制备的时间,减少了表达蛋白对细胞的损伤,提高了工作效率。
关于CSFV致病机制的研究报道甚少,本研究用猪瘟伪型病毒VSV△G*CSF感染敏感细胞PK-15不同的时间后,利用激光共聚焦显微镜观测E2蛋白在被感染细胞中的存在情况。分别对感染后10、20、35和60 min的PK-15细胞中E2的定位进行观测,结果表明:在被CSFV感染的过程中,E2蛋白先吸附在被感染细胞的细胞表面,逐渐进入到细胞内部,主要存在于细胞质中,不会进入到细胞核内,揭示了E2在CSFV的吸附与进入的过程中发挥关键作用。本研究为进一步研究猪瘟病毒的致病机制奠定基础。
虽然猪瘟伪型病毒研制成本较高,难以实现规模化大批量生产,同时VSV△G*CSF的病毒滴度较野生型CSFV低,但因为其生物安全性好,且携带有报告基因便于定性或定量检测,故可以作为中和试验中CSFV的替代品,成为新型检测工具应用于猪瘟中和抗体的测定,使CSFV的抗体检测更加安全,直观,快速。同时将猪瘟伪型病毒VSV△G*CSF作为引起主要保护性抗原E2基因的传递工具,用于研究被CSFV感染细胞中E2蛋白在病毒感染不同时间点的具体位置,动态的展现出E2蛋白从吸附到进入,以及感染剂量与感染时间的相互关系,为CSFV病毒感染细胞的研究提供参考。
Classical swine fever (CSF) caused by the Classical swine fever virus (CSFV) is an important contagious and fatal pig disease with widespread economic losses. Vaccination is of vital importance as an effective way to the prevention of CSFV infection, so it is essential to establish a stable and effective neutralizing antibody detection to test the CSF vaccine.
Pseudotype CSFV (VSV△G*CSF) refers to the process in which the enveloped protein gene (G) was replaced by the green fluorescent protein (GFP) gene and VSV△G* was formed, containing a new reporter gene. CSFV envelope protein from external sources can reassemble with VSV△G* and integrate into a one-off infectious VSV (Vesicular Stomatitis Virus) recombinant virus: VSV△G*CSF. VSV△G*CSF contains the genetic material of VSV and the capsule membrane integrated with the character of the infection of CSFV envelope protein. This kind of the inconsistency between genotype and phenotype is called pseudo-type, with such features of CSFV is called pseudotype virus of CSFV.
In this study, we constructed a recombinant eukaryotic expression plasmid pCAGG-E012 to express all of CSFV envelope proteins Erns, E1 and E2 by cis. According to the bovine viral diarrhea virus envelope glycoprotein E2 and to partially delete the different fragments by using the biology software to compare and analysis. The result of the expression indicted that after the cis expression of all CSFV envelope glycoproteins based on the recombinant eukaryotic expression plasmid pCAGG-E012, the titer of VSV△G*CSF is from 10~2 to 10~3·mL~(-1), and the infection can be neutralized with the positive serum anti CSFV.
The traditional methods for detecting CSF antibody like ELISA and indirect immunofluorescence assay have a series of flaws in urgent need of improvement, such as time-consuming, imprecise and low specificity. In order to improve the efficiency of neutralizing antibody detection and biological safety, and then to study the mechanism of CSFV infection, this study developed a replication-defective VSV/CSFV pseudovirus (VSV△G*CSF), as a model of CSFV infection to replace the wild-type CSFV virus as a novel neutralization assay to make the neutralization antibody detection process more convenient and accurate. The study shows that 200 pfu of VSV△G*CSFV could be neutralized by the standard positive CSF serum which is diluted 150 times. Using this method to test 10 serum samples, 7 were divided into positive and 3 were divided into negative, which have case detection rate of 90% compliance with that of commercial kit. Therefore, the method can be used as a new method in CSF antibody detection and has good application prospects.
Few reports are known for pathogenic mechanism of CSFV, some specific factors could significantly affect the results of CSFV infection, such as amino acid mutations in CSFV structural protein and changes of the major antigens E2 glycosylation site affect the virulence of CSFV and the combination of virus and target cells. The interaction of virus and cell is poorly understood, especially on the CSFV infection leading to host cell protein expression and biological function.
In this study, E2 protein locations on CSFV infected cells at different time point were discussed. CSFV infected PK-15 cells were detected by laser positioning confocal microscopy at 10, 20, 35 and 60 min, indicating that during the process of infected CSFV, E2 protein was attached to the cell surface of the infected cells, then entered the cell gradually, stayed in the cytoplasm, E2 do not enter the nucleus, so we can draw a conclusion that E2 protein played an important role in CSFV attachment and entry. This study provides a technical support for further study of CSFV infection mechanism.
Although the preparation of pseudo-type virus costs enormously, and it is difficult to achieve large-scale production at the same time, the titer of VSV△G*CSFV virus is lower than that of the wild type CSFV, it can be used as an alternative to the wild-type CSFV in neutralizing antibody detection, making the detection of CSFV antibodies more rapid, direct-viewing and secure, just because of its good bio-security and its ease of carrying the reporter gene for qualitative or quantitative analysis. On the other hand, pseudo-type virus VSV△G*CSFV can be used as a delivery tool of E2 gene to study the specific location of E2 protein in CSF infected cells at different time points, which can dynamically show the process that E2 protein from the adsorption to entry and the relationship between the infection dose and infection time. This result may be helpful for elucidating the CSFV infection mechanism.
引文
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