CN108048382A - A kind of six gene-deleted strain of actinobacillus pleuropneumoniae serum 1 type and its application without resistance marker - Google Patents

Abstract

The invention discloses an Actinobacillus pleuropneumoniae serotype 1 six-gene deletion strain without a resistance marker and an application thereof, belonging to the technical field of bacterial genetic engineering. The Actinobacillus pleuropneumoniae serotype 1 six-gene deletion strain of the present invention is an Actinobacillus pleuropneumoniae serotype 1 strain in which apxIC gene, apxIIC gene, apxIV-orf1 gene, cpxAR gene, arcA gene and ureA gene have been deleted. The strain does not contain resistance markers, has low virulence, is easily eliminated in the host body, and has good immune protection. It is an excellent attenuated vaccine and has important significance for the prevention and treatment of porcine infectious pleuropneumonia.

Description

A six-gene deficient strain of Actinobacillus pleuropneumoniae serotype 1 without resistance marker Lost plants and their applications

technical field

The invention belongs to the technical field of bacterial genetic engineering, and in particular relates to a six-gene deletion strain of Actinobacillus pleuropneumoniae serotype 1 without a resistance marker and an application thereof.

Background technique

Actinobacillus pleuropneumoniae (APP) is a Gram-negative bacterium belonging to the genus Actinobacillus of the Pasteurellaceae family (Pasteurellaceae). Mobility, can form spores. According to the dependence of APP growth on nicotinamide adenine dinucleotide (Nicotinamide Adenine Dinucleotide, NAD, also known as factor V), APP is divided into two biotypes, biological type I and biological type II. The growth of biotype I strains is NAD dependent, while the growth of biotype II strains is NAD independent. According to the classification of serotypes, it can be divided into 16 types. The serotypes prevailing in different places are different, and the cross-protection between different serotypes is not strong. Serum types 1, 2, 3 and 7 are mainly prevalent in my country, and all serotypes have different hemolytic activity and cytotoxicity.

Porcine contagious pleuropneumonia (PCP) is a porcine contagious respiratory disease caused by APP, which has caused serious economic losses to the world's pig industry. Since the prevalence of PCP was discovered in our country, the vast number of veterinary workers have conducted a series of researches on its etiology, epidemiology, diagnosis and control, and achieved certain achievements. But at present, the incidence of this disease in our country is still increasing year by year, and the positive rate of some pig farms has reached more than 70%, which has become one of the main infectious diseases in intensive pig farms, seriously endangering the pig industry of our country.

Antibiotics have played a great role in the prevention and control of bacterial infectious diseases, but with the frequent emergence of drug-resistant strains and the country's strict restrictions on the use of antibiotics, vaccines will become the main means of controlling porcine infectious pleuropneumonia. The new safe and efficient gene deletion vaccine will become a trend to control porcine infectious pleuropneumonia.

You Wujin et al. further deleted the arcA gene on the basis of the APP serum type 1 four-gene deletion strain CH04 (△apxIC/△apxIIC/△apxIV-orf1/△cpxAR) and obtained the APP serum type 1 five-gene deletion strain YWJ05 (△apxIC /△apxIIC/△apxIV-orf1/△cpxAR/△arcA), the five-gene deletion strain YWJ05 solved the problem of lung damage in the four-gene deletion strain CH04 (You Wujin, five-gene deletion vaccine strain of Actinobacillus pleuropneumoniae Construction and analysis of biological characteristics, master's degree thesis of Huazhong Agricultural University, 2013-06-01). However, the four-gene deletion strain CH04 and the five-gene deletion strain YWJ05 were less effective in being cleared by the body. A good vaccine needs to have lower virulence on the one hand, and better immune protection on the other hand, and it should be easily cleared by the body.

Contents of the invention

The purpose of the present invention is to overcome the defects and deficiencies in the prior art, and provide a more excellent attenuated vaccine strain—Actinobacillus pleuropneumoniae serotype 1 six-gene deletion strain without resistance marker. The object of the present invention is also to provide the application of the six-gene deletion strain.

The object of the present invention is achieved through the following technical solutions:

A six-gene deletion strain of Actinobacillus pleuropneumoniae serotype 1 that does not contain a resistance marker is a pig pleuropneumoniae that has deleted the apxIC gene, apxIIC gene, apxIV-orf1 gene, cpxAR gene, arcA gene and ureA gene Bacillus serotype 1 strain WBY06. The WBY06 strain is based on the five-gene deletion strain YWJ05 (You Wujin, Construction of Actinobacillus pleuropneumoniae five-gene deletion vaccine strain and analysis of its biological characteristics, master's degree thesis of Huazhong Agricultural University, 2013-06-01) to further delete the ureA gene get.

Further research on the biological characteristics of the WBY06 strain, such as virulence, tissue survival rate, and immune protection, found that the virulence of the WBY06 strain was significantly lower than that of the YWJ05 strain, and the WBY06 strain was easier to be eliminated in the body than the YWJ05 strain; the WBY06 strain was 3× The immune protection rate of mice challenged with 10 ⁸ CFU APP serum type 1 wild strain reached 100%. These results show that the WBY06 strain can be used to prepare porcine infectious pleuropneumonia vaccine.

A porcine infectious pleuropneumonia vaccine comprises the above-mentioned WBY06 bacterial strain.

The present invention mainly has following advantage and beneficial effect:

(1) The serotype bacterial strain used in the present invention is the dominant serotype that is prevalent in my country and seriously causes the disease of pigs at present, and has the strongest virulence and the best immune protection. Therefore, the vaccine made from the six-gene deletion strain constructed by the parent bacteria has strong pertinence for pig immunity and has broad market application prospects.

(2) Compared with the YWJ05 strain constructed by You Wujin et al., the WBY06 strain has lower virulence and is easier to be cleared in the body. It is a better attenuated vaccine and plays an important role in the prevention and treatment of porcine infectious pleuropneumonia. significance.

(3) The WBY06 bacterial strain of the present invention does not contain resistance markers, and fully meets the biological safety requirements of vaccines.

Description of drawings

Fig. 1 is a graph of the PCR identification results of the six-gene deletion mutant strain WBY06 of Actinobacillus pleuropneumoniae serotype 1. In the figure, M: DL2000, 1-4 is chloramphenicol gene detection PCR (primer cm-1/2), 1 is positive control plasmid pEMOC ₂ , 2 and 3 are WBY06 strain, 4 is ddH ₂ O negative control; 5 -9 is external primer verification PCR (primer pu1/2), 5 is ddH ₂ O negative control, 6 is pEM-ureA plasmid, 7 is positive control wild strain, 8, 9 is WBY06 strain.

Fig. 2 is a graph showing the experimental results of the growth characteristics of the WBY06 strain.

Figure 3 is a graph showing the results of lung colony counting.

Figure 4 is a graph showing the results of blood colony counts.

Detailed ways

The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. If not otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art

The six-gene deletion strain WBY06 of the present invention is based on the five-gene deletion strain YWJ05 constructed by You Wujin et al., and obtained by further deleting the urease gene ureA. The research results of YWJ05 have been published in the form of master thesis (You Wujin, Pleuropneumonia Construction of Actinobacillus five-gene deletion vaccine strain and analysis of biological characteristics, master's degree thesis of Huazhong Agricultural University, 2013-06-01). The present invention will be described in further detail below in conjunction with the embodiments and accompanying drawings. If not specified otherwise, the technical means used in the embodiments are conventional means well known to those skilled in the art.

Example 1 Construction of Actinobacillus pleuropneumoniae serotype 1 six-gene deletion strain WBY06

(1) Primer design

According to the reported sequence of APP-1 strain, two pairs of primers were designed to amplify the upstream and downstream arms of urease gene ureA. The sizes of the amplified fragments were 1019bp and 1037bp respectively. XbaI and NotI restriction sites were designed at the two ends of the arms respectively. The above primers were synthesized by Shanghai Bioengineering Company. The primer sequences are as follows:

Urea1: 5'-AGCGAATTCTTAAATTGCCTTTTACT-3'(SalI),

Urea2: 5'-GCTAAGCTTTGATTACTCTCAATCGC-3'(XbaI), upper arm 1019bp;

Urea3: 5'-GTACTCGAGCTAAGGAGACAACATG-3'(XbaI),

Urea4: 5'-ACACTCGAGGTTTGCTTACGCTCACG-3' (NotI), lower arm 1037bp.

(2) Cloning of the upstream arm and downstream arm of the urease gene ureA of Actinobacillus pleuropneumoniae

The improved TSA (i.e. tryptic soy agar medium) was purchased from GIBCO Company of the United States, with this medium as the basic component, additional 1% nicotinamide adenine dinucleotide and 10% calf serum by volume ) melted, cooled to 50°C, added an appropriate amount of nicotinamide adenine dinucleotide (NAD, purchased from Shanghai Chemical Reagent Company, China), poured it into a flat plate, and after cooling and solidifying, put it in a 37°C incubator for 2 to 3 hours until the water vapor drying. The lyophilized APP serotype 1 bacteria were inoculated into the drying plate for overnight culture. The next day, a single colony was picked and inoculated on modified TSB (i.e., tryptic soy agar medium, purchased from GIBCO Company of the United States, using this medium as the basic component, and adding 1% nicotinamide adenine dinucleotide by volume and 10% calf serum) culture medium at 37°C at 200r/min for 7-10h to extract the genome of the bacteria.

Take 1 mL of modified TSB culture-based EP tube, centrifuge at room temperature at 8000 r/min for 5 min, discard the supernatant, and suspend the pellet in 1 mL of LTE solution. Add 6 μL of 50 mg/mL lysozyme, act for 2 hours at 37°C, add 50 μL of 2mol/L NaCl, 110 μL of 10% sodium dodecylsulfonate (SDS), 3 μL of 20 mg/mL proteinase K, act for 3 hours at 50°C or 37°C overnight. Divide into two EP tubes equally, add equal volume of phenol: chloroform: isoamyl alcohol (100:99:1) to extract twice, precipitate with 0.6V isopropanol for more than 0.5h, centrifuge and then use 75% After washing with ethanol and drying in air, dissolve in 500 μL ddH ₂ O as a template for PCR.

The amplification reaction was carried out in a 50 μL system, and the reaction system was as follows: template DNA 2 μL, 10×PCR buffer 5 μL, 25 mmol/L MgCl ₂ 2.5 μL, 10 μmol/L upstream primer 0.5 μL, 10 μmol/L downstream primer 0.5 μL, 1 mmol /LdNTPs 2 μL, TaqE 1 μL, add ddH ₂ O to 50 μL.

Amplification conditions were as follows: denaturation at 95°C for 5 minutes, followed by cycling, and cycle parameters were 94°C for 1 min, 57°C for 1 min, and 72°C for 60 sec. After 35 cycles, extend at 72°C for 10 min. The amplified PCR product was analyzed by 0.8% agarose gel electrophoresis, and the sizes of the two amplified fragments were 1019bp and 1037bp, which were comparable to the expected size. The obtained target gene was cloned into the pMD-18 vector (purchased from Dalian Bao Biological Engineering Co., Ltd.), and sent to Dalian Bao Biological Engineering Co., Ltd. for the determination of the sequence of the exogenous gene.

(3) Construction of pEM-ureA transfer plasmid

The ureA upstream arm PCR amplification product with a size of 1019 bp was digested with SalI and XbaI, the ureA downstream arm PCR amplification product with a size of 1037 bp was digested with XbaI and NotI, and the conjugative transfer plasmid vector pEMOC2 was digested with SalI and NotI at the same time. Recover the upstream arm of ureA, the downstream arm of ureA and the vector pEMOC2, then connect them with T4DNA ligase, transform them into DH5α competent bacteria in a water bath at 16°C overnight, culture them at 37°C, pick the bacteria, and then transform the ligated products into DH5α Escherichia coli, and prepare plasmids in small quantities. Enzyme digestion and identification, thus obtaining the transfer plasmid pEM-ureA.

(4) Construction of ureA gene deletion strain WBY06 using YWJ05 as template

The constructed transfer plasmid pEM-ureA was transformed into Escherichia coli β2155, and then the Escherichia coli β2155 carrying pEM-ureA was transferred to the parental bacteria APP serotype 1 five-gene deletion strain YWJ05, and the chloramphenicol resistance plate ( Add 25 μg/mL of chloramphenicol into the TSA medium (sterilized at 50-60°C) to screen positive colonies, and culture and proliferate positive single colonies on TSB medium without chloramphenicol resistance to obtain resistance After colonization, sucrose-resistant colonies were screened on a 5% sucrose plate, and the ureA gene-deficient strain obtained through screening was named WBY06.

Genomic DNA of WBY06 strain was extracted, and the ureA gene was amplified by PCR with external primer pu-1/2 to determine whether a specific DNA fragment with a size of 518bp could be amplified. If the DNA fragment can be amplified instead of the same 2237bp fragment as the wild strain, it indicates that the result is in line with expectations, and the ureA gene deletion mutant strain bacterium that can be identified and screened is correct, and also utilizes cm-1 The /2 primer detects whether the mutant strain has a chloramphenicol resistance gene, and the experimental results are in line with expectations, as shown in Figure 1. The primer sequences used are as follows:

Pu-1: ATCAGTTGGCTGCCGAATA,

Pu-2: GAAACCAATCACGCCCTAA;

cm-1: TTTCAGGAGCTAAGGAAG,

cm-2: CACCAATAACTGCCTTAA.

Example 2 Growth characteristics and genetic stability analysis of the six-gene deletion strain WBY06

Pick a single colony of wild bacteria (APP1, WT) and the six-gene deletion strain WBY06, inoculate them into TSB medium for culture, take samples every 1 hour, and read their values with a spectrophotometer OD ₆₀₀ , and pass through each same time period The OD ₆₀₀ value was used to compare their growth speed. The results are shown in Figure 2: Compared with the APP serotype 1 wild strain, the six-gene deletion strain WBY06 only grew slower in the logarithmic phase, and there was no difference in other time periods.

Passage the six-gene deletion strain WBY06 on TSA medium, sample once every other generation, and continuously passage for more than 20 times. Randomly select several generations, and use the external primer pu-1/2 for PCR amplification. Only the size can be amplified. It is a specific DNA fragment of 518bp, indicating that the six-gene deletion strain WBY06 can be inherited stably.

Example 3 Evaluation of the safety and immune protection of the six-gene deletion strain WBY06

(1) Toxicity test in mice

The safety of WBY06 strain was evaluated by measuring the median lethal dose LD ₅₀ of Balb/c mice. Seventy-two 6-week-old Balb/c mice were equally divided into 12 groups, with 6 mice in each group. Among them, 0.2mL of wild bacteria (APP1, WT) was injected intraperitoneally into each mouse in the 4 groups, and the infection doses were 2.40×10 ⁷ CFU, 1.20×10 ⁷ CFU, 6.00×10 ⁶ CFU, 3.00×10 ⁶ CFU; Each mouse was intraperitoneally injected with 0.2mL five-gene deletion strain YWJ05, and the infection doses were 1.66×10 ⁹ CFU, 1.00×10 ⁹ CFU, 6.02× ^{10 8} CFU, and 3.62×10 ⁸ CFU; each mouse in 4 groups was injected intraperitoneally For 0.2mL six-gene deletion strain WBY06, the infection doses were 9.05×10 ⁹ CFU, 5.40×10 ⁹ CFU, 3.25×10 ⁹ CFU, and 1.95×10 ⁹ CFU. Continuously observe for 10 days, record the death rate of the mice, and evaluate the virulence of the six-gene deletion strain WBY06. The median lethal dose of mice was calculated according to the Reed-Meunch method.

The results are shown in Table 1: the LD ₅₀ of wild bacteria is 9.50×10 ⁶ CFU, the LD ₅₀ of YWJ05 is 9.10×10 ⁸ CFU, and the LD ₅₀ of WBY06 is 5.40×10 ⁹ CFU. The results of LD ₅₀ showed that the virulence of the WBY06 strain was much lower than that of the wild strain; and the LD ₅₀ of the WBY06 strain _was 6 times that of the five-gene deletion strain YWJ05, which indicated that the virulence of the WBY06 strain was also much lower than that of the YWJ05 strain. It is a safer attenuated vaccine candidate strain.

Table 1 The LD ₅₀ of the six-gene deletion strain WBY06 prepared by the present invention to Balb/C mice

(2) Tissue survival test

Twenty-seven 6-week-old Balb/c mice were equally divided into 3 groups, with 9 mice in each group. The APP serotype 1 wild strain, YWJ05 strain, and WBY06 strain were inoculated intraperitoneally into mice at a dose of 3.00×10 ⁶ CFU, respectively. 24h, 72h, and 120h after inoculation, 3 mice were dissected from each group, and 0.1g of lungs were taken in 1mL PBS, homogenized with a homogenizer; the blood was directly diluted. Do multiple dilutions with PBS, spread the dilutions of different dilutions on the TSA plate, place in a constant temperature incubator at 37°C and cultivate overnight, and count when a single colony grows. The results are shown in Figures 3 and 4. At different time points after inoculation, the number of WBY06 strain in lung and blood was smaller than that of YWJ05 strain, indicating that WBY06 strain was easier to be cleared by the body than YWJ05 strain.

(3) Eighteen 6-week-old Balb/c mice were divided into 3 groups on average, 6 mice in each group, namely YWJ05 test group, WBY06 test group and control group. In the YWJ05 test group, 200 μL of the bacterial solution containing 3×10 ⁸ CFU YWJ05 was injected intraperitoneally, in the WBY06 test group, each mouse was injected with 200 μL of the bacterial solution containing 3×10 ⁸ CFU WBY06, and in the control group, each mouse was injected with 200 μL of PBS. The second immunization was carried out 14 days after the first immunization, and the dose of the second immunization was the same. Fourteen days after the second immunization, the mice were challenged with 3×10 ⁸ CFU of APP serum type 1 wild bacteria, and the clinical manifestations and death of the mice were observed and recorded every day after the challenge. On the second day of the challenge, all 6 mice in the control group died; after 14 days of continuous observation, no mice died in the YWJ05 test group and WBY06 test group, and the immune protection rates of the YWJ05 test group and WBY06 test group reached 100%.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

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Claims (3)

1. a kind of six gene-deleted strain of actinobacillus pleuropneumoniae serum 1 type without resistance marker, it is characterised in that：It is scarce The pig chest of apxIC genes, apxIIC genes, apxIV-orf1 genes, cpxAR genes, arcA genes and ureA genes is lost Film Actinobacillus serum 1 type bacterial strain.

2. application of six gene-deleted strain described in claim 1 in porcine contagious pleuropneumonia vaccine is prepared.

3. a kind of porcine contagious pleuropneumonia vaccine, it is characterised in that：Include six gene-deleted strain described in claim 1.