Recombinant Porcine Rotavirus VP6 Proteins Expressed in Lactobacillus plantarum NC8 Strain Induces Specific Mucosal and Systemic Antibody Production in Experimental BALB/c mice

Rotaviruses are the main cause of animal and infant diarrhea and are widely distributed worldwide. In the pig industry, porcine rotavirus infection is a significant cause of mortality and morbidity; therefore, the optimization and well-organized distribution of vaccines for infection prevention is needed. Because immune responses related to protection are mainly mucosal in nature, the induction of mucosal immunity is significant for preventing porcine rotavirus infection. The major protective VP6 antigens against porcine rotavirus (PRV) expressed by Lactobacillus plantarum NC8-pSIP409-pgsA-VP6-Dcpep were used to orally immunize mice. Western blot analysis and SDS-PAGE were used to confirm the expression of recombinant NC8-pSIP409-pgsA-VP6-Dcpep, and immunofluorescence was also used to verify its surface expression on L. plantarum NC8. plantarum for oral vaccinations and the performance of L. plantarum as an antigen delivery system. Following the administration of live bacteria to mice, the immunogenic ability of these recombinant strains was analyzed. The oral administration of the recombinant strain NC8-pSIP409-pgsA-VP6-Dcpep stimulated specific anti-rotavirus systemic and mucosal immune responses. In mice immunized with L. plantarum NC8 expressing the VP6-Dcpep fusion, the effectiveness of the immune response evaluation was greater than in mice immunized with NC8-pSIP409-pgsA, indicating the effectiveness of Dcpep as a mucosal adjuvant. Using standard molecular biology techniques, the recombinant NC8-pSIP409-pgsA-VP6-Dcpep was constructed to recognize DCs and the VP6-antigen more efficiently in the intestinal mucosa, and its immunogenicity was further investigated. The results indicated that the in vivo antibody levels were substantially improved after BALB/c mice were immunized by the lavage administration of our recombinant NC8-pSIP409-pgsA-VP6-Dcpep.


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NC8 is effective and that Dcpep is superior in its ability to stimulate mucosal immunity, suggesting that this approach can be implanted in pigs.

Background
Rotaviruses are members of the family Reoviridae. Depending on their particular inner capsid protein sequences, porcine rotaviruses are categorized into groups A, B and C [1].
Rotaviruses have a nonenveloped, double-stranded RNA genome that is composed of 11 segments enclosed by a triple-layered icosahedral capsid, [2][3][4][5]. The main source of acute diarrhea in piglets is porcine rotaviruses, which can cause high rates of mortality and morbidity [6][7][8][9][10][11]. In both pre-and post-weaning pigs, rotavirus Group A is the main source of rotavirus-associated diarrhea and accounts for 54% and 45% of the diarrhea experienced in those populations of pigs, respectively [1]. Some research has reported that in commercial pig farms, 89% of all rotavirus-associated diarrhea can be attributed to group A rotavirus infections [12]. Rotavirus outbreaks are difficult to prevent because they are transmitted via the fecal oral route and can survive in the environment for a long period of time. Replication of the virion takes place in the intestinal villi in epithelial cells and destroys enterocytes mainly in the ileum and jejunum, leading to villous atrophy [13][14][15][16]. In addition, in the affected regions, nutrients cannot be absorbed or digested, causing severe malabsorption [13][14][15][16]. The control of rotavirus infections requires a solid understanding of the epidemiology of rotaviruses, which will contribute to prevention programs and improve the current vaccines. Presently, the obtainable vaccines cannot provide adequate protection. To activate immunocompetence, repeated administrations and a large vaccine dose are generally needed. This repeatedly causes unwanted clinical signs. To overcome these weaknesses and deliver antigens to the mucosal immune system, possible improvements to lactic acid bacteria (LAB) have been proposed.
Mucosal immunity plays an important role in protective immunity because rotaviruses are 4 enteric pathogens. In the gut, innate immune responses initiate acquired immune responses and offer the first line of protection against pathogenic microorganisms. In addition, the only appropriate way of eliciting gut immunity is to prompt the immune response via oral immunization because this oral route assists in gut-associated lymphoid tissue (GALT) stimulation, improving anti-viral IgA production [17,18].
Live vaccines stimulate the most efficient defensive responses, unlike heat-killed or recombinant antigen formulations, because they elicit both mucosal and systemic immunity [17,18]. Repeated vaccinations and large vaccination doses are needed due to the challenges posed by oral vaccination, such as the fact that the gut environment habitually deactivates and/or denatures potential vaccinogens, causing fever and diarrhea, with the live vaccine often being shed in the feces [17,18]. With regard to the stimulation of mucosal immunity, lactic acid bacteria (LAB) can be used to overcome these challenges [19]. Furthermore, several LAB strains are capable of colonizing and surviving the intestinal tract, stimulating nonspecific immunoadjuvant consequences [19]. It is essential to boost the immunogenicity of genetically engineered vaccines by combining them with suitable adjuvants because they are poorly immunogenic and composed of a single recombinant antigen. The dendritic cells play a central role in targeting peptide by directing innateand regulating adaptive/acquired immunity.By phage display the three DCbinding 12 number of residues in the peptide 12-mer: pep3 FYPSYHSTPQRP, pep12 AYYKTASLAPAE and pep18SLSLLTMPGNAS was recognized in 2004. Out of the three peptide pep3 later was renamed Dcpep, for the reason that Dcpep bound to both immature and mature DCs in a saturable way, than pep12 or pep18 and had the fascinating property to bind both human Monocyte-Derived Dendritic CellsmdDCs and mouse CD11c+ I-A+ DCs [20] Herein, we constructed recombinant L. plantarum NC8 strains expressing porcine rotavirus 5 VP6 to test the effectiveness of the expression of the VP6 porcine rotavirus protein by L. plantarum for oral vaccinations and the performance of L. plantarum as an antigen delivery system. Following the administration of live bacteria to mice, the immunogenic ability of these recombinant strains was analyzed. The oral administration of the recombinant strain NC8-pSIP409-pgsA-VP6-Dcpep stimulated specific anti-rotavirus systemic and mucosal immune responses. In mice immunized with L. plantarum NC8 expressing the VP6-Dcpep fusion, the effectiveness of the immune response evaluation was greater than in mice immunized with NC8-pSIP409-pgsA, indicating the effectiveness of Dcpep as a mucosal adjuvant. Using standard molecular biology techniques, the recombinant NC8-pSIP409-pgsA-VP6-Dcpep was constructed to recognize DCs and the VP6antigen more efficiently in the intestinal mucosa, and its immunogenicity was further investigated. The results indicated that the in vivo antibody levels were substantially improved after BALB/c mice were immunized by the lavage administration of our recombinant NC8-pSIP409-pgsA-VP6-Dcpep.

Results
Cloning, expression and purification of Rotaviral target genes VP6 in

Prokaryotic Expression System
The recombinant plasmids were identified by restriction endonuclease digestion enzyme Xba I and Hind III digestion. As shown in Fig. 1, two distinct bands appeared which were in accordance with the expected sizes. The results of sequencing were compared by NCBI Blast, and the homology was 100%.
To confirm VP6 expression, the recombinant NC8-pSIP409-pgsA-VP6-Dcpep was SppIP-6 induced and cultured. Anti-VP6 serum from rabbits was used as an antibody; IgG from goats labeled with FITC was used as the secondary fluorescent antibody. FlowJo 7.6.1 software was used for detection. The results showed that the peak value of NC8-pSIP409-pgsA-VP6-Dcpep group was 92.7%, compared with 57.3% count in the NC8-pSIP409-pgsA group, as shown in Fig. 2 The results showed that compared with the empty carrier group, the content of IL-4 in the NC8-pSIP409-pgsA-VP6-Dcpep group was significantly greater (P < 0.001). There was also significant difference (P < 0.001) in IFN-gamma between the NC8-pSIP409-pgsA-VP6-Dcpep group and the blank carrier group (Fig 6).

Discussion
The main sources of acute diarrhea in piglets are porcine rotaviruses, and infection with these viruses can lead to mild to severe diarrhea with high mortality and morbidity rates.
Porcine rotavirus infection has been an economic concern worldwide among pig breeders. was obtained from previous work [27] in which it was used for the heterologous expression of target genes (VP6 and NC8) in a prokaryotic expression system.

Mice
In this model, we used Rotavirus antibody-free adult female Balb/C mice that were 7 weeks of age (weighing 25-30 g) obtained from Beijing Huafukang Biotechnology Co., Ltd.
China; these mice are used to investigate the immune responses to the recombinant NC8- an Xba I site. The PCR was carried out in a standard 50 ml reaction for the amplification of the VP6 gene using the primer sets following standard protocols. The tubes were then placed in a thermocycler, and the PCR conditions for amplification were as follows: predenaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 cycles and extension at 72°C for 10 minutes. After the reaction was finished, the 2 L PCR product was electrophoresed on a 0.8% agarose gel.
Electroporation of L. plantarum was carried out as follows: 10 μl of plasmid was gently added to 100 μl of L. plantarum NC8, mixed gently for 5 min at 4°C and subjected to an 14 electric pulse. The mixture of plasmid DNA and L. plantarum NC8 was then anaerobically incubated without EM in MRS medium for 2 h at 37°C. Recombinant NC8-pSIP409-pgsA-VP6-Dcpep was selected on MRS agar medium including EM. The transformants with L. plantarum NC8 sequences were verified by plasmid DNA sequencing.

Flow cytometry
The cells were carefully washed with PBS, centrifuged at 12000 rpm for 5 min at 4°C, and washed twice with PBS; then, 10 μL of cells were added to each flow cytometry tube and incubated at room temperature for 20 min in the dark. The cells were washed twice with cold PBS, centrifuged at 4°C and 2000 rpm for 5 min; the cell suspension was centrifuged, and approximately 300 μL was transferred to a flow tube. The effect of lactic acid bacteria on the activation B cells was detected by flow cytometry. The data were analyzed using FlowJo 7.6.1 software [28].
Protein expression and sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blot analysis NC8-pSIP409 and NC8-pSIP409-pgsA-VP6-Dcpep frozen at -80°C were subcultured. The second generation of bacterial liquid was added to 50 mL of MRS liquid medium (Em+) at a 1:100 ratio. Anaerobic culture was carried out in a 30°C incubator until the OD value was 0.3. SppIP was added at a ratio of 1:400 for overnight induction. The overnight culture solution was centrifuged for 10 minutes at 4°C and 5000 g, and the supernatant was discarded. The supernatant was washed three times with cold PBS. The precipitation was suspended in 500 µL of pyrolysis buffer. The supernatant was digested at 120 rpm for 30 minutes in a shaker at 37°C and centrifuged at 10000 g for 30 minutes. When the supernatant was collected, the soluble cell wall component, VP6, was contained in the supernatant. A total of 160 µL was removed from the supernatant and added to 40 µL of the 5 x SDS buffer. The protein was denatured by boiling at 100°C for 5 minutes. The supernatant was centrifuged for 2 min at 12 000 rpm. SDS-PAGE was performed.

SDS-PAGE
Two plates of glue were allocated for SDS-PAGE, one of which was dyed and decolorized according to the above method and observed on the machine; the other was carried out according to the following method.

Transmembrane
After the SDS-PAGE was finished, the gel was removed, and transfer filter paper was soaked in transfer buffer for 30 min. The PVDF membrane was first activated for 30 s with methanol and then soaked in the transfer buffer. According to the size of the gel, the filter paper and PVDF film were cut and then placed in the transfer buffer. The order was sponge, 3 layers of filter paper, PVDF film, gel, 3 layers of filter paper and sponge. The bubbles generated between each layer were removed by glass rods and then the gel was placed into the transfer clamp. The gel side was connected with the negative electrode.

Enzyme-linked immunosorbent assay (ELISA)
To examine specific anti-VP6 antibodies, mouse serum was collected on days 14, 35 and 42 after immunization and centrifuged at 4000 rpm for 15 min at 4°C to obtain the serum, which was stored at -80°C and then analyzed by ELISA as described previously [29]. ELISA was performed to determine the levels of specific IgG and sIgA antibodies [30]. Feces samples were also collected at 14, 35 and 42 days after immunization. The serum samples (1:100) and fecal samples (1:10) were added separately after dilution and, following incubation for 2 h at 37°C, the wells were rinsed 5 times. Then, goat anti-mouse IgA-Biotin and goat anti-mouse IgG (H+L)-Biotin (Southern Biotechnology, Birmingham, AL) were added separately, and the plate was incubated at 37°C for one hour, after which streptavidin-HRP (Southern Biotechnology, Birmingham, AL) secondary antibody was added and the plate was incubated at 37°C for 10 min. Finally, chromogen solution A and B and stop solution were added, after which the color intensity was measured at 450 nm.
The end-point titers (log 2 ) were defined as the highest dilution yielding an absorbance that was two and three times higher than the background for the serum and fecal samples, respectively [31].

Detection of cytokines
The serum samples were examined for specific cytokines, including IFN-γ and IL-4, using an ELISA kit (LIUHEBIO, Wuhan, China) according to the manufacturer's instructions. Statistical analysis Flowjo 7.6.1 software was used for flow cytometry analysis, GraphPad Prism 5 software was used for mapping and statistical data, and one-way ANOVA was used for statistical analysis of the differences between groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001). This study was carried out in agreement with the principles established by Jilin Agriculture University Changchun China and guide for the use of laboratory and care animals and all experimental protocols were approved by a Jilin Agriculture University (No. JLAU08201007).

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Competing Interests:
Author1 declares that she has no conflict of interest.
Author 2 declares that he has no conflict of interest.
Author 3 declares that she has no conflict of interest.
Author 4 declares that he has no conflict of interest    Histopathological changes in mice infected with Chinese porcine rotavirus isolate DN30209 strains following immunization

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