Salmonella choleraesuis attenuated recombinant strain as well as construction method and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses a construction method of a swine cholera attenuated salmonella recombinant strain capable of simultaneously expressing a haemophilus parasuis outer membrane protein GAPDH gene and an Omp26 gene, a plasmid connected with the haemophilus parasuis GAPDH gene and the Omp26 gene is adopted to transfect a swine cholera salmonella asd gene deleted strain, screening is carried out, and screening is carried out to obtain the swine cholera attenuated salmonella recombinant strain capable of simultaneously expressing the haemophilus parasuis outer membrane protein GAPDH gene and the Omp26 gene. The recombinant attenuated salmonella choleraesuis expressing the haemophilus parasuis GAPDH gene and the Omp26 gene is obtained. The strain can simultaneously express a haemophilus parasuis outer membrane protein GAPDH gene and an Omp26 gene, and compared with a strain which singly expresses Omp26, the strain has the advantage that the immune protection effect is obviously improved. Meanwhile, the invention also provides a construction method and application of the strain.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a recombinant strain of attenuated Salmonella choleraesuis and a construction method and application thereof. Background Art

[0002] Haemophilus parasuis (HPS) can clinically cause HPS disease characterized by multiple fibrinous serositis, pericarditis and arthritis. In recent years, the prevalence of the disease has been increasing, and it has become one of the important bacterial diseases in pig production. At present, inactivated vaccines are widely used in clinical practice, but they cannot provide complete cross-immunity protection due to the existence of different serotypes. The research and development of subunit vaccines mainly focuses on virulence factors such as outer membrane proteins, capsular polysaccharides, and lipopolysaccharides. Among them, GAPDH is a membrane protein of HPS. GAPDH is an enzyme involved in the glycolysis pathway and is involved in the virulence and pathogenicity of HPS; and Omp26, as a type of HPS outer membrane protein, is one of the main virulence factors of HPS and has good immunogenicity. However, conventional subunit vaccines usually express only one protein, which may lead to low immunogenicity. In addition, studies have shown that recombinant bacterial vector vaccines can present antigenic proteins to the host in a similar way to natural infection, inducing the host to produce an immune response, and have a stronger immune effect than recombinant subunit vaccines. Moreover, the way in which recombinant bacterial vector vaccines present antigenic proteins to the host immune system after immunizing animals is similar to natural infection, and can induce the host to produce cellular immunity, humoral immunity, and mucosal immunity.

[0003] Infectious diseases of pigs caused by Salmonella are also widely prevalent in pig farms in my country, which has had a certain impact on my country's pig farming industry and production. In the current context of reducing antibiotics and replacing antibiotics, it has become difficult to use antibiotics to prevent and treat bacterial infectious diseases. Now studies have shown that the research idea of ​​transforming Salmonella and preparing it into a live carrier of attenuated vaccines has shown broad application prospects. Studies have shown that the asd gene is required for the synthesis pathway of DAP, the main component of Gram-negative bacteria cells. The deletion of the asd gene will cause the mutant strain to die under the condition of no exogenous DAP. Only bacteria containing asd+ plasmids can survive in hosts without exogenous DAP. The pYA3493 plasmid carrying the asd gene of Salmonella typhimurium was selected for exogenous gene expression, which can be balanced with the C500Δasd strain.

[0004] The applicant previously disclosed CN109652414A to construct a recombinant attenuated Salmonella choleraesuis strain expressing the Omp26 gene of Haemophilus parasuis, by constructing the asd gene-deficient strain C500Δasd of Salmonella choleraesuis; then electrotransferring the recombinant vector into the asd gene-deficient strain C500Δasd of Salmonella choleraesuis, followed by culturing, to obtain the recombinant attenuated Salmonella choleraesuis strain C501Δasd pYA3493-Omp26 expressing the outer membrane protein gene Omp 26 of Haemophilus parasuis. The strain can express the Omp26 gene. The applicant previously disclosed CN109652413A to construct a recombinant attenuated Salmonella choleraesuis strain expressing the GAPDH gene of Haemophilus parasuis, construct the pYA3493-GAPDH recombinant vector through EcoRI and Hind III restriction sites, and then electrophoretically transfer the recombinant vector into the asd gene deleted strain C500Δasd of the Salmonella choleraesuis, followed by culturing to obtain the Haemophilus parasuis outer membrane protein gene GAPDH recombinant attenuated Salmonella choleraesuis strain C501ΔasdpYA3493-GAPDH.

[0005] However, the immune protection ability of the strains mentioned above is not strong, and the strains need to be further modified to obtain strains with good protective effects. Summary of the invention

[0006] The object of the present invention is to provide a recombinant attenuated Salmonella choleraesuis strain, which can simultaneously express the outer membrane protein GAPDH gene and Omp26 gene of Haemophilus parasuis, and has significantly improved immune protection effect compared with strains that only express Omp26 and strains that only express GAPDH.

[0007] At the same time, the present invention also provides a construction method and use of the strain.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The invention discloses a method for constructing a recombinant strain of attenuated Salmonella choleraesuis that simultaneously expresses the outer membrane protein GAPDH gene and Omp26 gene of Haemophilus parasuis. The asd gene-deficient strain of Salmonella choleraesuis is transfected with a plasmid connected with the GAPDH gene and the Omp26 gene of Haemophilus parasuis. After screening, the recombinant attenuated Salmonella choleraesuis that expresses the GAPDH gene and the Omp26 gene of Haemophilus parasuis is obtained.

[0010] In the above construction method, the Haemophilus parasuis is Haemophilus parasuis type 5 SH0165 strain.

[0011] In the above construction method, the plasmid includes a vector and the GAPDH gene and Omp26 gene connected to the vector; the vector is the pYA3493 plasmid.

[0012] Meanwhile, the present invention also discloses a recombinant strain of attenuated Salmonella choleraesuis, which is prepared by any of the above methods.

[0013] At the same time, the invention also discloses the use of the attenuated Salmonella choleraesuis recombinant strain for preparing vaccines.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention connects two important immunogenic genes GAPDH and Omp26 of Haemophilus parasuis to the pYA3493 vector to construct a recombinant vector pYA-GAPDH-Omp26, and then electrotransfers the successfully constructed vector into the asd gene-deficient strain C500Δasd of Salmonella choleraesuis, thereby screening and constructing a recombinant vaccine strain, which not only expresses the two antigen genes of Haemophilus parasuis but also carries the antigen gene of Salmonella choleraesuis. The recombinant attenuated Salmonella choleraesuis C500Δasd / pYA-GAPDH-Omp26 strain constructed by the present invention has genetic stability after detection; the growth characteristics are not significantly different from those of the control bacteria; and the effect is significant in the level of induced antibodies, and good protection can be provided after secondary immunization. The research results of the present invention lay a foundation for the subsequent research and development of the combined vaccine of choleraesuis and Haemophilus parasuis.

[0016] In the preparation method of the present invention, corresponding primers are designed for two surface antigens GAPDH and Omp26 of Haemophilus parasuis, and target fragments are obtained by PCR; GAPDH and Omp26 are connected to pY3493 plasmid by enzyme cutting and connection to construct a recombinant vector pYA-GAPDH-Omp26, and the recombinant plasmid is electrotransferred into the host bacteria C500Δasd to construct an attenuated Salmonella choleraesuis strain that simultaneously expresses GAPDH and Omp26 genes, i.e., C500Δasd / pYA-GAPDH-Omp26; the genetic stability and growth characteristics of the recombinant strain are analyzed, and it is found that the growth characteristics are similar to those of the parent strain and can be stably inherited. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the electrophoresis diagram of the target fragment GAPDH amplified by PCR;

[0018] Figure 2 This is the electrophoresis diagram of the target fragment Omp26 amplified by PCR;

[0019] Figure 3This is a graph showing the stability of the recombinant strain C501pYA3493-GADPH-Omp26;

[0020] Figure 4 This is a growth curve of the recombinant strain C501pYA3493-GADPH-Omp26. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1: Referring to the gene sequences of membrane protein GAPDH (GenBank ID: 72785966) and outer membrane protein Omp26 (GenBank ID: 219691637) of Haemophilus parasuis serotype 5 strain SH02165 in Genebank, primers were designed using Primer5.1, and the genome of the standard Haemophilus parasuis type 5 strain was used as a template to amplify GAPDH containing EcoRI and Sal I restriction sites, and Omp26 containing Sal I and Hind III restriction sites, respectively.

[0023] GAPDH primer sequence 1: 5′-CGGAATTCATGGCAATTAAAATTG-3′;

[0024] GAPDH primer sequence 2: 5′-GCGTCGACTTAGCCTTTGTAGTTG-3′;

[0025] Omp26 primer sequence 3: 5′-GCGTCGACATGAAAAATTTATTTAAACTTGC-3′;

[0026] Omp26 primer sequence 4: 5′-CCAAGCTTTTATTTTTTCACTTCTTCTGG-3′.

[0027] Example 2

[0028] The recombinant vector was constructed, DNA of Haemophilus parasuis type 5 strain SH0165 was extracted, and the primer pair in Example 1 was used to amplify the Omp26 gene and the GAPDH gene, respectively.

[0029] The PCR amplification conditions of Omp25 gene were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min; annealing at 56°C for 1 min; extension at 72°C for 1 min, for 32 cycles.

[0030] The PCR amplification conditions of GAPDH gene were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 45 s; annealing at 55°C for 30 s; extension at 72°C for 1 min 30 s, for 34 cycles.

[0031] The amplified PCR product was recovered by the gel recovery kit instructions. The electrophoresis of the PCR amplified target fragment GAPDH can be seen Figure 1 The electrophoresis diagram of the target fragment Omp26 amplified by PCR shows Figure 2 ; Figure 1 and Figure 2 Clear bright strips of the target fragment can be seen in the image.

[0032] The obtained fragment was connected with the pMD-18T vector, and the ligation product was transferred into competent cells DH5α (purchased from TaKara), ice-bathed for 30 minutes, heat-shocked at 42°C for 1 minute, and ice-bathed for 2 minutes; then 650 μL of LB liquid culture medium was added, shaken at 220 r / min for 1 hour, and centrifuged at 3000 rpm / min for 3 minutes. A portion of the supernatant was discarded, and the bacteria were resuspended in the remaining culture medium, spread on a solid plate (containing 100 μL / mL Amp+), and cultured at 37°C for 12 hours.

[0033] A single colony was picked and inoculated into 3mL LB (containing 50μg / mL Amp+) liquid medium, shaken at 220rpm / min for 12h, and the plasmid was extracted using a kit. After restriction digestion, screening and sequence determination, it was named pMD18-T-GAPDH and pMD18-T-Omp26. The pYA3493 plasmid and pMD18-T-GAPDH were double-digested with EcoR I and Sal I to construct the pYA-GAPDH vector; then the pMD18-T-Omp26 and pYA-GAPDH vectors were double-digested with Sal I and Hind III, and introduced into Escherichia coli x6097 competent cells after ligation with T4 ligase, and the pYA-GAPDH-Omp26 plasmid, i.e., the recombinant vector, was screened and identified by Shanghai Biotech sequencing.

[0034] Take 10μL of pYA-GAPDH-Omp26 plasmid and add it to 100μL competent cells C500Δasd, mix gently and transfer to a pre-cooled 0.2cm electroporation cup, dry the water outside the electroporation cup, and transform the voltage to 2.0KV, the capacitance is 5uF; the pulse resistance is 200Ω, and the time is 4ms. After the electric shock, take out the electroporation cup and add 1.5mL LB medium, 37 degrees 220r / min, and culture for 1h. Take out the bacterial solution and spread it on a solid plate at 37℃ for 12h. Until a single colony grows, pick a single colony for PCR identification and sequencing, and the results all prove that the positive recombinant strain C501pYA3493-GADPH-Omp26 is obtained.

[0035] Example 3

[0036] The recombinant strain C501pYA3493-GADPH-Omp26 was continuously subcultured in a non-resistant LB liquid medium and identified until the 100th generation. GAPDH primer 1 and Omp26 primer 4 were used for amplification and identification to determine whether the exogenous gene could stably exist in the host bacteria.

[0037] refer to Figure 3 , Figure 3 From left to right in the figure are the amplification diagrams of the GAPDH target fragments of the 1st, 5th, 10th, 25th, 50th, 75th and 100th generations. It can be seen that when it is passed down to the 100th generation, the exogenous gene can still stably exist in the host bacteria.

[0038] Example 4

[0039] The obtained recombinant strain C501pYA3493-GADPH-Omp26 was streaked on a non-resistant plate, and single clones of about 0.5 mm were picked and inoculated into LB liquid culture medium. After shaking culture at 37°C for 10 h, the bacterial solution value was adjusted to be consistent, and then 50 μL of bacterial solution was inoculated into liquid culture medium and shaken at 37°C. 200 μL of bacterial solution was taken out every 1 h to measure the OD 562 The growth curve of the recombinant strain was drawn with the culture time of the bacterial solution as the horizontal axis and the average value of the bacterial solution as the vertical axis.

[0040] refer to Figure 4 , Figure 4 is a comparison diagram of the growth curves of the recombinant strain C501pYA3493-GADPH-Omp26 and the parent strain pYA3493; Figure 4 It can be seen that there is almost no difference in the growth curves of the two.

[0041] Example 5

[0042] The mice were divided into two groups. One group was challenged with the virulent HPS SH0165 strain of Haemophilus parasuis type 5 (5LD50) by intraperitoneal administration at a dose of 500 million / 0.2 mL / mouse. The other group was challenged with the virulent Salmonella choleraesuis strain C78-1 by oral administration at a dose of 5LD50, i.e. 5 million / 0.1 mL / mouse.

[0043] Before oral administration, mice were fasted for 24 hours and deprived of water for 6 hours, and gastric acid was neutralized with 100 μL 10% NaHCO3. The vaccine (recombinant strain C501pYA3493-GADPH-Omp26) was then administered orally with a No. 12 oral injection needle. After the challenge, drinking water and feed were restored. The mice were observed for 14 consecutive days after the challenge, and the clinical characteristics and mortality of each group of mice were recorded.

[0044] For details, please refer to Table 1 and Table 2;

[0045] Table 1: Protection against Salmonella choleraesuis C78-1 in immunized mice

[0046]

[0047] Table 2: Protection of immunized mice against Haemophilus parasuis

[0048]

[0049] The above experiments show that the recombinant strain C501pYA3493-GADPH-Omp26 of the present invention can simultaneously express the outer membrane protein GAPDH gene and Omp26 gene of Haemophilus parasuis, and its protection against Salmonella choleraesuis C78-1 and Haemophilus parasuis is significantly improved.

Claims

1. A method for constructing a recombinant strain of attenuated Salmonella choleraesuis that simultaneously expresses the outer membrane protein GAPDH gene and Omp26 gene of Haemophilus parasuis, characterized in that: The plasmid connected with GAPDH gene and Omp26 gene of Haemophilus parasuis was used to transfect the asd gene-deficient strain of Salmonella choleraesuis, and the recombinant attenuated Salmonella choleraesuis expressing GAPDH gene and Omp26 gene of Haemophilus parasuis was obtained through screening.

2. The construction method according to claim 1, characterized in that: The Haemophilus parasuis is the Haemophilus parasuis type 5 SH0165 strain.

3. The construction method according to claim 1, characterized in that: The plasmid comprises a vector and a GAPDH gene and an Omp26 gene connected to the vector; the vector is a pYA3493 plasmid.

4. A recombinant strain of attenuated Salmonella choleraesuis, characterized in that: The method is prepared by any one of claims 1 to 3.

5. Use of the attenuated Salmonella choleraesuis recombinant strain as claimed in claim 4 in preparing a vaccine.

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