NS1 is essential for viral DNA replication, as well as the VP2 proteins is the main antigenic domain that may induce PPV-neutralizing antibodies and play an integral function in the prevention and medical diagnosis of PPV [5]

NS1 is essential for viral DNA replication, as well as the VP2 proteins is the main antigenic domain that may induce PPV-neutralizing antibodies and play an integral function in the prevention and medical diagnosis of PPV [5]. result in a blended infection, straight leading to sow mating obstructions [4]. This disease causes huge economic losses to the pig breeding industry [5,6,7]. Nowadays, infections caused by PPV are very serious and have spread all over the world, and the positive antibody rate of swine exceeds 90%. PPV is a small, non-enveloped and single-stranded DNA virus, which belongs to the genus and the family [8]. The genome of PPV consists of two large open reading frames, which encode non-structural protein NS1 and several capsid proteins, such as VP1, VP2, and VP3 [9,10]. NS1 is necessary for viral DNA replication, and the VP2 protein is the major antigenic domain that can induce PPV-neutralizing antibodies and play a key role in the prevention and diagnosis of PPV [5]. Previous studies have demonstrated that the recombinantly expressed VP2 protein can self-assemble into virus-like particles, which have ideal immunogenicity and can stimulate the body to produce high antibody titers. Classical inactivated PPV vaccine is available commercially and widely used to control PPV infections Anacardic Acid [11,12]. However, the safety and multiple immunizations with large doses are the major considerations of the inactivated vaccine, leading to the development of the PPV subunit vaccine. The adjuvant is an indispensable component of a subunit vaccines to improve the immunogenicity because it can induce stronger immune responses and reduce the dosage and production cost. A better adjuvant can not only enhance the immunogenicity of the vaccine and activate the immune system, such as cytotoxic T lymphocytes (CTLs) or helper T lymphocytes (THs), but also strengthen the humoral and/or cellular immune responses [13]. Natural polysaccharides are characterized by intrinsic immunomodulation, biocompatibility, biodegradability, low toxicity and safety, and a Anacardic Acid variety of natural polysaccharides have been proved to have better immune promoting effects and can enhance immune responses [14]. Among the natural polysaccharides, chitosan is a biopolymer of cationic polysaccharides with lots of biological properties, including low toxicity, good biocompatibility, and biodegradability [15,16], while the weak water solubility of chitosan greatly limits its application. One of the strategies to improve the solubility of chitosan is to modify the molecular structure by the addition of hydrophilic functional groups [17]. Our laboratory has synthesized the water-soluble AcNPV-VP2, sf21 (cell system and purified using Ni-NTA affinity column chromatography. The Anacardic Acid purified VP2 protein was separated by SDS-PAGE and then electro-transferred onto nitrocellulose membrane for Western blotting analysis. The membranes were blocked with 5% skim milk in TBST buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05% Tween 20, pH 7.5) at 4 C overnight; Subsequently, the membranes were incubated with swine anti-PPV serum obtained from the Harbin Veterinary Research Institute at a dilution of 1 1:1000 with shaking overnight at 4 C. The membranes were then washed three times with TBST buffer, each time for 5 min, followed by incubation with horseradish peroxidase-conjugated rabbit anti-pig IgG antibody (Sigma, St. Louis, MO, USA) for 1 h, and then the membranes were washed with TBST buffer three times. The immunoreactive bands were visualized by a Licor ODYSSEY near-infrared fluorescent scanning imaging system (Licor, Lincoln, NE, USA). 2.3. Preparation of the PPV/VP2/N-2-HACC Hemagglutination (HA) titer of the VP2 protein was tested by HA assay [25]. The VP2 protein solution was mixed with the N-2-HACC IKZF2 antibody solution at a ratio of 0.5% ( 0.05 was considered statistically significant. 3. Results 3.1. Expression of the PPV VP2 Protein In Vitro Figure 1 shows that the PPV VP2 protein was purified. To confirm the expression of the VP2 protein, the purified VP2 protein was analyzed by Western blotting analysis using PPV-positive pig serum, and the polyclonal antibodies recognized VP2 protein with a molecular weight of 64 KDa (Figure 1), and the densitometry readings of target bands were 10,717 using the Image J software (NIH, Bethesda, MD, USA). Open in a separate window Figure 1 Western blotting analysis of the PPV VP2 expressed in sf21 cells. M: Protein ladder; 1: Precipitation of cells (the purified VP2); 2: Supernatant of the cell culture. To confirm the PPV VP2 expression, the purified VP2 was subjected to Western blotting analysis. The primary antibody is PPV-positive pig serum, and the secondary antibody is horseradish peroxidase-conjugated rabbit anti-pig IgG antibody. 3.2. Sterility of the PPV/VP2/N-2-HACC The prepared PPV/VP2/N-2-HACC was milky white liquid and easily detached from the bottle wall by gently shaking, and the lower layer was slightly reddish after.