Cloning and expression of PirA gene of Vibrio parahaemolyticus strain K5 causing acute hepatopancreatic necrosis disease in whiteleg shrimp in E. coli host cell

Background: Acute hepatopancreatic necrosis disease (AHPND), is a bacterial disease of whiteleg shrimp, which has a high mortality rate (100%) and incurs economic losses. Our objective was to identify the genes which lead to cell and organ damage and investigate bioproducts to prevent and treat. Methods: Litopenaeus vannamei shrimp in Thua Thien Hue province, Vietnam were collected from an infected pond and analysed at the Institute of Biotechnology, Hue University. The PirA gene of Vibrio parahaemolyticus strain K5 was isolated and analyzed for nucleotide sequence and paired with the expression vector pQE30. The expression vector was transformed into E. coli strain M15, the PirA recombinant protein was expressed in the form of 6xHis-PirA fusion protein of about 15 kDa. PirA recombinant PirA gene from Vibrio parahaemolyticus strain K5 causing AHPND by PCR method with specific primers and molecular weights of PirAvp and the PirAvp complex. Results: PirA gene from Vibrio parahaemolyticus strain K5 was cloned into pGEM-T easy vector (Promega, USA) and screened E. coli TOP10 colonies containing pGEM T easy/PirA recombinant plasmid on LB agar/ampicillin/IPTG/X-Gal medium. PCR showing a band of about 347 bp, matching the size of PirA gene and two nucleotide sequences (BamHI and HindIII). The results showed that PirA gene has a length of 336 bp and similar to PirA gene on GenBank (Code: KU556825.1). The results of protein extracted from E. coli M15 recombinant cells and 6xHis-PirA target protein was collected in elution fractions from EF2 to EF6, showed that the concentration of 6xHis-PirA protein and EF3 elution fraction collected a highest protein concentration (1,586.54 µg/ml). Conclusions: The purified PirA recombinant protein will provide materials for development research to create biological products to prevent and treat AHPND. GoTaq® Green Master Mix (Promega, USA) and 4 μl of nuclease-free water for the amplication of the pirA gene. The reactions were run on MJ MiniTM Personal Thermal Cycler (BioRad, USA). PirA gene was amplified using the following parameters: initial denaturation at 95°C for 5 minutes, 30 cycles of 95°C for 30 seconds, 53°C for 30 seconds and 72°C for 1 minute 30 seconds, followed by a final extension at 72°C for 10 minutes and store at 4°C. The PCR was with Tukey’s test at a probability level of P ≤ 0.05.

Thien Hue (Vietnam) with clinical signs of AHPND (N. N. Quang, et al., 2013). Dao et al. (2014) identified some characteristics of Vibrio parahaemolyticus strain V1 isolated from juvenile whiteleg shrimp showing clinical signs of AHPND in Thua Thien Hue. The study revealed it is Gram-negative bacterium, rod shaped, 0.3 -0.5 µm in width and 1.4 -2.6 µm in length. In addition, its colonies are mauve, round, smooth with a darker center than the surrounding on CHROM agar medium (Dao, et al., 2014). V. parahaemolyticus is mainly distributed in marine and estuarine environments around the world (Wang et al., 2015). The strains of AHPND pathogenic bacteria contain an intracellular plasmid, which is not found in non-pathogenic strains (Xiao et al., 2017). This large plasmid (69-70 kb) contains PirAvp and PirBvp toxin genes (Han, Tang, Tran, & Lightner, 2015;Yang et al., 2014) that encode PirABvp binary protein, which has been shown to be the major virulence factor (Han, Tang, Tran, et al., 2015;Lee et al., 2015;Sirikharin et al., 2015). Quang et al. (2020) isolated 14 strains of Vibrio spp.
carrying PirAvp and PirBvp genes from whiteleg shrimp samples cultured in Tam Giang lagoon (Vietnam) (Quang et al., 2020). From whiteleg shrimps infected AHPND in Phong Dien district, Thua Thien Hue province (Vietnam), we isolated V. parahaemolyticus strain K5 carrying both PirA and PirB toxin genes with predicted size of 336 bp and 1,317 bp, respectively (Khanh et al., 2019) In this study, we sequenced the PirA gene of the isolated V. parahaemolyticus strain K5, created E. coli

Cloning and sequencing of PirA gene
The PirA gene was isolated from Vibrio parahaemolyticus strain K5 causing AHPND on whiteleg shrimp by PCR method with specific primers. A shotgun sequencing of bacterial small subunit ribosomal DNA gene fragments amplified from AHPND infected shrimp revealed that the bacterial sequences of VpAHPND strains were not related to sequences normally found in diseased shrimp infected with other Vibrio bacterial species. A phylogenetic analysis showed that the strains were genetically diverse and not derived from a single genetic lineage. On the other hand, a preliminary sequence assembly and comparison analysis of the strains suggested that the target sequences originated from plasmid. The identified contigs of the strains were found to be homologous not to chromosomes of known V. parahaemolyticus but to the contigs obtained from other VpAHPND strains. Furthermore, a contig encodes the homologues of type IV pilus protein and conjugal transfer protein in the strain was also revealed, which suggests that it is located on a plasmid to insert the PirA gene into the pQE30 expression vector, specific primers BamHI and HindIII restriction enzymes were added (BamHI_PirA_1F forward primer: 5'-GGATCCATGAGTAACAATATAAAACATG-3'; HindIII_PirA_336R reverse primer: 5'-AAGCTTAGTGGTAATAGATTGTACAG-3').
PCR was carried out in 12 μl reactions containing 1 μl of genomic DNA template, 1 μl of each primer (10 pmol), 6 μl of 2X GoTaq® Green Master Mix (Promega, USA) and 4 μl of nuclease-free water for the amplication of the pirA gene. The reactions were run on MJ MiniTM Personal Thermal Cycler (BioRad, USA). PirA gene was amplified using the following parameters: initial denaturation at 95°C for 5 minutes, 30 cycles of 95°C for 30 seconds, 53°C for 30 seconds and 72°C for 1 minute 30 seconds, followed by a final extension at 72°C for 10 minutes and store at 4°C. The PCR product was electrophoresed on a 1% agarose gel, voltage of 80 V in 1X TAE buffer with SafeView dye (1X TAE: SafeView = 20:1). Electrophoresis result was observed on Ultra Slim LED Illuminator (Miulab, China).
The PCR product was cloned in pGEM®-T easy vector and transformed into E.coli TOP10 host cells by heat shock method (Froger & Hall, 2007). The E. coli were grown for 24 to 36 h on LB (NaCl 10 g/L, Yeast extract 5 g/L, Peptone 10 g/L) agar medium supplemented with 100 µg/ml ampicillin + 100 mM IPTG + 200 mg/ml X-Gal (LB agar/ampicillin/IPTG/X-Gal medium) and the best grown were selected. Five white colonies were randomly selected and PCR with BamHI_PirA_1F and  (Froger & Hall, 2007). Five transformed colonies grown on LB agar supplemented with 100 g/ml ampicillin and 50 g/ml kanamycin were randomly selected and further cultured in liquid LB medium supplemented with 100 g/ml ampicillin and 50 μg/ml ampicillin for recombinant plasmid  (Bradford, 1976).

Data analysis
Data were statistically analyzed using Minitab software version 16.2.0 and Microsoft Excel 2013 to calculate the mean and standard deviation. ANOVA was used to identify significantly different means compared between elution fractions with Tukey's test at a probability level of P ≤ 0.05.

Sequencing of PirA gene
Amplification product of PirA gene from Vibrio parahaemolyticus strain K5 was cloned into pGEM-T easy vector (Promega, USA). We screened E. coli TOP10 colonies containing pGEM T easy/PirA and PirA gene sequence from Vibrio parahaemolyticus strain K5 is 100% similar to PirA gene from Vibrio parahaemolyticus strain V.03 which has been published on GenBank (accession number: KU556825.1).

Creation pQE30/PirA recombinant plasmid
The PirA gene and pQE30 vector were collected and purified from digestion products by BamHI and HindIII restriction enzymes, after pQE30 vector and PirA gene were paired by the enzyme T4 DNA ligase to create pQE30/PirA recombinant plasmid. The coupling product was then transformed into E.
coli strain TOP10 and first screened on LB Agar medium supplemented with 100ug/ml ampicillin. The transformation result showed that white colonies appeared on LB agar/ampicillin medium (Figure 2A).
This indicates a successful transformation, and these colonies may contain pQE30/PirA recombinant plasmid. We randomly selected five colonies for biomass culture and plasmid extraction. PCR was performed with the extracted DNA plasmid template, and BamHI_PirA_1F and HindIII_PirA_336R primers to confirm the presence of PirA gene. The PCR product was electrophoresed on 1% agarose gel ( Figure 2B).   that the majority of 6xHis-PirA fusion protein was collected in elution fractions from EF2 to EF6, while most of the basic proteins of E. coli M15 that was unable to bind to Ni 2+ ions on the HisTrap FF column were removed during wash step with the binding buffer (20 mM sodium phosphate, 0.5 M NaCl, 5 mM imidazole) previously. Most of the protein 6xHis-PirA binding on the column was collected from EF2 to EF5 elution fractions, while 6xHis-PirA protein was collected very little at EF1 and EF6 elution fractions. Thus, the 6xHis-PirA protein has been purified successfully. However, there is still a small amount of impurity protein in the purified product in EF1 and EF2 elution fractions.
Analytical results by Bradford method (Table 1) showed that the concentration of 6xHis-PirA protein in elution fractions from HisTrap FF column was different. In which, EF3 elution fraction collected the highest target protein concentration (1,586.54 µg/ml), followed by EF2 elution fraction (1,317.1 µg/ml).
The collected protein concentrations decreased gradually from EF3 to EF6 elution fractions, reaching the lowest in EF6 (17.46 µg/ml). The means with different letters within the same row are significantly different between elution fractions at the 0.05 probability level.

Discussion
The parahaemolyticus strain 13-028/A3 showed that the toxin proteins were encoded by two 2 genes (pirAlike and pirB-like) in a segment 3.5 kb, the pirA-like gene (336 bp) and the pirB-like gene (1,317 bp) encoded for 13 kDa and 50 kDa proteins, respectively .
Our research results showed that the expression of PirA gene was produced in the form of a fusion protein with high concentration. The molecular weight of this fusion protein was approximately 15 kDa (including 6xHis tag of pQE30 vector) (Figure 3). This size is higher than found in study by Han et al. (2015), where proteomic analysis showed that the pirA-like genes (336 bp) encode for a protein with the size of 13 kDa . Lee et al. (2015) performed western blot analysis with antibodies against PirA vp from AHPND causing V. parahaemolyticus and found that this strain secreted PirA vp (12 kDa) into the culture medium after 1 h after cultivation (Lee, et al., 2015). The variation in the size of the PirA protein may be due to the influence of several factors during SDS PAGE electrophoresis and the effect of the expression system on PirA gene expression.
The PirA antigen was expressed by pQE30 expressing vector system and was fused with six amino acids histidine. This 6xHis tag has a high affinity for Ni2 + ions on the Histrap FF column (GE Healthcare, USA), allowing for easy collection of 6xHis-PirA fusion protein and removal of other proteins of E. coli M15 cell when their total proteins are passed through the chromatographic column.
The 6xHis-PirA fusion protein recovered from the Histrap FF column had high purity and concentration ( Figure 4 and Table 1), and can be used as a raw material for further research to produce antibodies to prevent AHPND in shrimp caused by Vibrio spp.

Competing interests
No competing interests were disclosed.

Grant information
This investigation was funded by the Ministry of Education and Training, Vietnam, through a ministrylevel science and technology project (reference: CT2018-DHH-04).