加热法促进猪圆环病毒2型病毒样颗粒的高效组装

Heat Treatment Promotes Efficient Assembly of Porcine Circovirus Type 2 Virus Like Particles

为了提高猪圆环病毒2型(PCV2)衣壳(Cap)蛋白病毒样颗粒(VLPs)的产量,本试验探索了采用加热法促进杆状病毒-昆虫细胞表达的Cap蛋白体外组装的可行性,通过高效液相尺寸排阻色谱法定量PCV2 Cap VLPs,分析4~56℃加热0~10 h对细胞培养液上清中PCV2 Cap VLPs浓度的影响,并采用透射电子显微镜(TEM)验证;通过监测45℃加热6 h后的细胞培养液上清在4℃放置6个月中PCV2 Cap VLPs的浓度变化,验证PCV2 Cap VLPs的稳定性;通过全能核酸酶消化试验验证宿主核酸对PCV2 Cap VLPs组装的作用;对比加热与未加热工艺的PCV2 Cap VLPs纯化收率,并通过高效液相尺寸排阻色谱偶联多角度激光散射、圆二色光谱、差示扫描量热法和微量热泳动分析2种工艺所得PCV2 Cap VLPs表征的一致性。结果显示,细胞培养液上清中PCV2 Cap VLPs浓度随着温度和时间的延长逐渐升高,其中45℃加热6 h后PCV2 Cap VLPs浓度为未加热的3.77倍,TEM观察进一步验证PCV2 Cap VLPs浓度增加。加热后形成的PCV2 Cap VLPs在4℃储存6个月浓度稳定,未出现解聚现象。全能核酸酶消化试验显示,加热过程中宿主核酸的作用并非关键因素。采用45℃加热6 h后再进行纯化,PCV2 Cap VLPs纯化收率是未加热工艺的2.9倍。经分析,加热与未加热工艺所得PCV2 Cap VLPs具有一致的分子量(2.385×10^(6)和2.361×10^(6) Da)、水力学半径(10.1和10.2 nm)、热转变温度Tm(67.22和66.92℃)、二级结构和特异性抗体亲和力(49.0和76.5 pmol/L),表明两者具有相近结构。本试验为PCV2 Cap VLPs的生产探索了一个提高产量的新方法,为兽用疫苗的研发提供了在抗原性质分析方法上的借鉴。

To increase the yield of porcine circovirus type 2(PCV2)capsid(Cap)protein virus like particles(VLPs),this study explored the feasibility of promoting the assembly of Cap protein expressed by the baculovirus-insect cell system using heat treatment method.High-performance size-exclusion chromatography was used to quantify PCV2 Cap VLPs,analyzing the effects of heating from 4℃ to 56℃ for 0-10 hours on the concentration of PCV2 Cap VLPs in the cell culture supernatant,and verified using transmission electron microscopy(TEM).The stability of PCV2 Cap VLPs was confirmed by monitoring concentration changes after heating at 45℃ for 6 hours and storing the supernatant at 4℃ for 6 months.Benzonase digestion test was conducted to verify the role of host nucleic acids in the assembly of PCV2 Cap VLPs.The purification yield of PCV2 Cap VLPs was compared between the heated and unheated processes,and consistency in characterization between the two methods was analyzed using high performance size exclusion chromatography-multi-angle laser light scattering,circular dichroic spectrum,differential scanning calorimetry,and microscale thermophoresis.The results showed that the concentration of PCV2 Cap VLPs in the cell culture supernatant gradually increased with rising temperature and extended heating time.Specifically,heating at 45℃ for 6 hours resulted in PCV2 Cap VLPs concentration 3.77 times higher than the unheated control.TEM observations further validated the increased PCV2 Cap VLPs concentration.The formed PCV2 Cap VLPs after heating remained stable at 4℃ for 6 months,showing no signs of disaggregation.Benzonase digestion test indicated that host nucleic acids were not a critical factor during heating.After purification,the yield of PCV2 Cap VLPs from the heated process was 2.9 times higher than that of the unheated process.Characterization analysis revealed consistent molecular weights(2.385×10^(6) Da and 2.361×10^(6) Da),hydrodynamic radii(10.1 nm and 10.2 nm),melting temperatures(T m of 67.22℃ and 66.92℃),secondary structures,and specific antibody affinities(49.0 pmol/L and 76.5 pmol/L)between the two processes,indicating similar structures.This study presents a new method for enhancing PCV2 Cap VLPs production,providing insights into the analysis of antigen properties for veterinary vaccine development.