藏猪白细胞介素-12基因表达对猪圆环病毒2 疫苗的免疫增强效应

Elevation of immunity to PCV2 vaccine by recombinant Tibetan pig interleukin-12 gene in chitosan nanoparticles

摘要:

为研究白细胞介素-12（IL-12）基因在猪体内表达对圆环病毒2型（PCV2）疫苗免疫应答的调控作用，克隆了藏猪IL-12的P40和P35基因，并将完整的IL-12基因克隆至真核表达载体VR1020，经体外表达验证后，采用离子交联法对藏猪IL-12基因重组质粒进行包裹，制备成粒径小、分散性好且带正电荷的壳聚糖纳米颗粒（VRIL-12-CNP），与PCV2疫苗共同肌肉注射接种21日龄断奶仔猪，在接种后第0天、7天、14天和28天时采集猪前腔静脉血进行血常规分析、流式细胞分析、血清抗体检测和相关免疫基因表达量检测，并记录其生长体重。结果发现：接种了VRIL-12-CNP的实验组猪血液中的CD3+ T细胞、Th细胞、Tc细胞和PCV2抗体在接种后都显著增长（P<0.05）；并且实验组TLR2、TLR7、IL-12、IL-4、IL-6和IL-15基因的表达量都显著高于对照组（P<0.05）；实验组STAT1、STAT3和Bcl-2基因表达水平也同样显著增高（P<0.05）；实验组猪的体重净增长在试验期间也明显高于对照组（P<0.05）。这些结果充分表明：VRIL12-CNP不仅能增强PCV2疫苗的先天免疫应答，也能增强体液免疫和细胞免疫应答，表明VRIL-12-CNP是一种能安全、高效地增强PCV2疫苗免疫应答的免疫佐剂。

Abstract:

To study the regulation effect on vaccination of porcine circovirus-2 (PCV2) by co-delivering interleukin-12 (IL-12) cDNA via chitosan nanoparticles, two subunits of IL-12, P40 and P35 of Tibetan pig, were cloned into mammalian expression plasmid VR1020. VRIL-12 was transfected into HEK293 cells and PCR test was conducted. The results showed that IL-12 gene could express successfully. The recombinant plasmid was packaged into chitosan nanoparticles using ionic crosslinking method. The nanoparticles (VRIL-12-CNP) were confirmed to be nanoparticle size with good dispersion and positive charge. VRIL-12-CNP was then used to inoculate the 21-day-old piglets together with PCV2 vaccine. Blood samples were taken at day 0, 7, 14 and 28 post inoculation for analysis. The results showed that post vaccination the T cells such as CD3+, CD4+ , CD8+, and anti-PCV2 specific antibody, all increased significantly in the VRIL-12-CNP co-delivering pigs (P<0.05), the expression levels of TLR2, TLR7, IL-12, IL-4, IL-6 and IL-15 in the co-delivering group were significantly higher than those in control group (P<0.05), and the expression of STAT1, 3 and Bcl-2 gene was also increased significantly in the co-delivering pigs (P<0.05). Moreover, the net weight gain of the pigs in co-delivery group was significantly increased in the 28 days post inoculation (P<0.05). These results indicated that co-delivery of VRIL-12-CNP elevated swine innate immune response and both humoral and cellular immune response to PCV2, and VRIL-12-CNP is a promising safe and effective adjuvant to PCV2 vaccination.