Construction Of Apx â…¡ Genetic Engineering Mutant Strain Of Actinobacillus Pleuropneuminiae And Research On Its Biological Characteristic

Porcine contagious pleuropneumonia (PCP) is an infectious porcine respiratory tract disease causing severe economic losses worldwide in the swine industry, caused by Actinobacillus pleuropneumoniae (APP).Immunization is the main method to prevent and control this disease. The current commercial vaccines are still primarity killed whole cell bacterins and subunit vaccines, which generally reduce mortality from APP infection but frequently fail to prevent serever morbidity and economic loss due to the chronic effects of the disease on growth rate and feed efficiency.In contrast, natural and experimental infection can induce protection against any heterologous serotype. Thus, live attenuated vaccines might be a viable approach to solve this problem. The previously described methods for the construction of mutant strains of A. pleuropneuminiae have some disadvantage. For example, mutagenesis using a chemical or a transposon mediated approaches present a random approach and allow for the selection of mutants with a distinct phenotype only; muttions not resulting in a distinct phenotype can not be constructed by these means. All these mutants , which is achieved by systems allowing targeted mutagenesis by homologous recombination, however, carry a permanent antibiotic resistance marker and, therefore, are not suitable as vaccine strains. In order to construct the genetic engineering mutant strain not carrying antibiotic resistance determinant, So the following researches were explored.1. Identification of the omlA gene promoter sequence of A pleuropneuminiaeThe chromosomal DNA of A. pleuropneuminiae serotype l(S4074), for which the transcriptional start site of omlA gene had been determined, was used as template for a single 157 bp omlA gene promoter sequence amplification by polymerase chain reaction (PCR). The DNA fragment was further cloned and the complete sequences were determined. The resulting fragment was cloned into upstream from the promoterless GFP gene encoding the green fluorescent protein in the plasmid pGFP-2. The resulting plasmid pMSKOGFP, carrying the 157 bp omlA promoter sequence directed toward the GFP gene was transformed into A pleuropneuminiae. To determine the ability of the omlA promoter sequence to support GFP expression, Ampicillne resistant A. pleuropneuminiae transformants were tested for green fluorescence by fluorescent microscopy. The results showed that the 157 bp omlA promoter sequence can promote GFP gene expression.2. Characterization of sacB gene expression in A. pleuropneuminiaeThe sacB gene expression leads to bacterial death in the presence of sucrose. The sacB gene encoding the enzyme levansucras .which drived from the Bacillus subtillus,was amplified from the plasmid_PGS284 by PCR and cloned into pMSK-omlA to generate recombinant plasmid pMSKOS containing the omlA gene promoter sequence in the same orientation as the sacB gene. To characterize sacB expression in A. pleuropneuminiae, the plasmid pSKOS containing omlA gene promoter sequence and pSKS not containing omlA gene promoter sequence was transformed into A. pleuropneuminiae serotype 7 strain by electrotransformation. The Ampicillin resistant transformants were analyzed for sucrose sensitivity in a TSA plate containing 5% sucrose. The growth of transformants carrying plasmid pMSKOS was inhibited completely while the growth of transformants carrying plasmid pMSKS was only slightly inhibited. The results demonstrated that APP was motality when the omlA gene promoter sequence promote sacB gene expression in APP and the sacB gene can be used for conterselection marker to construct APP mutant strains..3. Cloning and sequence analysis of apx Ⅱ CA geneThe apx II gene consist of the apx Ⅱ C gene encoding posttranslational activator, and apx Ⅱ A gene encoding the structural toxin. When the apx Ⅱ C gene was inactivated, virulence of the strain was decreasing obviously, but the expression of ApxⅡA still occurs in this strain. In our work, the complete DNA encoding Apx II protein of APP strain HB serotype 7 was amplified by polymerase chain reaction (PCR). The DNA fragment was further cloned and the complete sequences were determined. The results indicate that the DNA of apx ⅡCA encodes 1054 aa residues. Compared with the apx ⅡCA gene of APP 1, 2, 3 and 7 serotypes strain, it was found that there was over 99% aa identity.4. Construction of recombinant transfer plasmid pSK-x ⅡCA-/GSA+The amplified apxⅡCA gene was digested with PstI and ligated to the PstI linearized pBluescripts II sk(+) vector to obtain plasmid pSK-apx Ⅱ CA. A 720bp fragment encoding GFP was amplified from plasmid pGFP-2 and inserted at the site-specific Xba Ⅰ site in the apx ⅡC gene to form plasmid pSK-XIICA-/GFP+. Then, a 157 bp promoter sequence of the outer membrane lipoprotein (omlA) from the APP genome, the promoterless sacB gene encoding levansucrase from plasmid PGS284, and the ampicillin resistance gene from plasmid pCI-neo, were amplified by PCR with appropriate restriction sites incorporated at the primers. Their sequences were confirmed by sequencing, and ligated to create an omlA-sacB-Ampr cassette in pBluescripts II sk(+). The cassette was ligated to linearize pSK-X Ⅱ CA-/GFP+ by NotI and SmaI vector, and yield recombinant transfer plasmid pSK-X ⅡCA-/GFP+.5. Selection and evaluation of the genetic engineering mutant strain HBC-/GFP+.Plasmid pSK-xIICA-/GSA+was introduced into A. pleuropneumoniae competentcells and ampicillin resistant integrants were selected. Fifty colonies were replica-plated to TA-sucrose (TA-Suc) plates in order to confirm sucrose sensitivity. All of them were Ampr, Sucs colonies and three were selected for further analysis. In these strains, pSK-x ⅡCA-/GSA+ was integrated via homologous recombination. Cells were grown overnight in TSB in the absence of antibiotic selection to promote recombination and sucrose-resistant clones were isolated by plating cells on TA-Suc in the appropriate dilution in order to have approximately 100 colonies per plate. Two hundred Sucr colonies were replica plated to TA-Suc-Amp and TA-Suc plates in order to choose the Amps ones. Finally, 8 mutants were selected that could grow on plates with 5% sucrose (Suer) but could not grow on plates containing 5% sucrose and amplicillin (Amps). The one mutants were confirmed as having specific gene disruption by PCR and Southern blotting by showing insertion of the GFP gene in the apx ⅡC gene region. One representative Amps Sucr strain, named A. pleuropneumoniae HBC-/GFP+. The genetic engineering mutant strain HBC-/GFP+was verified and evaluated by PCR, Southern blot. Then, Its some biological activities such as hemolytic activity, growth rate, immunitical activity (Western blot), the genetic stabily of the insertional mutants were more investigated. 6. Safety of the genetic engineering mutant strain HBC-/GFP+ in Balb/C mice and pigletsTo test the virulence of the genetic engineering mutant strain HBC-/GFP+, various concentrations of A. pleuropneumoniae HBC-/GFP+ and APP serotype 7 strains were prepared and administrated to mice via the intraperitoneal(i.p) route. No deaths were observed in mice that received at doses of 2×108, 1×109 and 2×109 CFU, whilst 100% mortality was recorded in mice that received the parent strain at doses of 2×107, 1×108 and 2×108 CFU. Therefore, compared to the parent strain, virulence of the genetic engineering mutant strain HBC-/GFP+ is safety for mice.To test the safety of the genetic engineering mutant strain HBC-/GFP+ in piglets, 1×108 CFU and 2×108 CFU dilutions of the genetic engineering mutant strain HBC-/GFP+ were prepared in bacterial growth medium and administered to piglets via intranasal. In the first day after injection, there are some clinical signs of pleuropneumonia such as increased respiration rate, labored breathing (dyspnea), decreased appetite and activity/attitude (lethargy) in piglets. The temperature of piglets administered the genetic engineering mutant strain HBC-/GFP+ is only slightly increasing. However, these clinical signs was disappeared the second day after injection. The results showed that the genetic engineering mutant strain HBC-/GFP+ is safety for piglets.7. Vaccination and challenge efficacy of the the genetic engineering mutant strain HBC-/GFP+ in Balb/C mice and pigletsTo evaluate the vaccination efficacy of the genetic engineering mutant strain HBC-/GFP+, mice were vaccinated with 2×108 CFU of HBC-/GFP+ strain via inoculation intraperitoneally on days 0 and 14. Antibodies were detected with ELISA and IHA at 14 and 28 days after first immunitation. The results showed that antibodies level of Apx Ⅱ-ELISA and IHA immunized with the genetic engineering mutant strain HBC-/GFP+ were more higher than those of killed vaccine. In addition to, antibody level of Apx Ⅱ -ELISA and IHA increased obviously after second immunization. Two weeks after secondary vaccination, the immunized mice groups together with TSB and killed vaccine control groups were challenged with 2.0 ml containing 1×109 CFU of A. pleuropneumoniae serotype 1 and 2×109CFU serotype 7. Killed vaccine offered 50% (4/8) and 75% (6/8) against serovar 1 and 7 respectively, while the genetic engineering mutant strain HBC-/GFP+ offered 75% (6/8) and 100% (8/8) against serovar 1 and 7, respectively.To test the protective efficacy of the genetic engineering mutant strain HBC-/GFP+ for piglets, piglets were vaccinated twice via intranasal inoculation with 1×109CFU of the genetic engineering mutant strain HBC-/GFP+ at 2-week internals. The antibody of each antigen was detected with ELISA and IHA. Piglets were challenged two weeks after second vaccination by intranasal injection with 2.0ml containing 1×109CFU of APP serotype 1 and 2.0 ml containing 2×109CFU of APP serotype 7, respectively. The protective efficacy of immunization was evaluated by clinical, bacteriological, serological and post-mortem examination. The results demonstrated that antibody level of Apx Ⅱ -ELISA and IHA increased obviously after second immunization. Antibody level of Apx Ⅱ-ELISA and IHA immunized with the genetic engineering mutant strain HBC-/GFP+ were more higher than those of killed vaccine. Piglets vaccinated via intranasal inoculation with the genetic engineering mutant strain HBC-/GFP+ had 100% and 75% protection against homologous serotype (serotype 7) and heterologous serotype (serotype 1) challenge with A. pleuropneumoniae, respectively. But, killed vaccine only offered 75% and 50% protection against homologous (serotype 7) and heterologous (serotype 1) challenge with A. pleuropneumoniae, respectively. The results suggested that immunization with the genetic engineering mutant strain HBC-/GFP+ could alleviate clinical sign and lung lesion, which was more efficacious than killed vaccine, and the genetic engineering mutant strain HBC-/GFP+ is a kind of potential APP vaccine strain.