oahs gene deletion strain and its application in preventing streptococcus suis type 2 infection

Abstract

The application belongs to the technical field of genetic engineering, and provides an oahs gene deletion strain and application thereof in prevention of Streptococcus suis type 2 infection.The oahs gene deletion strain, namely SC19-ΔOAHS, has very low virulence and is basically non-toxic.The mutant strain has basically no infection damage to mice when the mice are immunized with a large dose of live bacteria, and the antibody titer in the mice is obviously increased with the aid of an adjuvant, so that the immunoprotection can be successfully established.The immunoprotection established after a normal immunization procedure can help the mice effectively resist the infection of Streptococcus suis type 2, reduce the mortality and bacterial colonization in the body.The preparation method of SC19-ΔOAHS is simple, and the strain can be prepared in a short period of time, is simple to culture, and does not need other complex treatment as a vaccine, and can be used after washing and mixing with an adjuvant.SC19-ΔOAHS has the potential to be used as a live bacteria vaccine against Streptococcus suis infection in pigs.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and in particular relates to oahs gene deletion strains and their application in the prevention of Streptococcus suis type 2 infection. Background Technology

[0002] Streptococcus suis (SS) is a Gram-positive opportunistic bacterium that colonizes the upper respiratory tract asymptomatically. Based on capsular polysaccharide antigens, it is classified into 29 different serotypes, with SS type 2 being the most pathogenic and having the highest clinical isolation rate. SS can cause life-threatening diseases, including meningitis, pneumonia, arthritis, endocarditis, and acute septicemia. Therefore, this pathogen seriously threatens the global swine industry and public health security. An early outbreak of inflammatory cytokines can lead to streptococcal toxic shock-like syndrome (STSLS) within 13 hours of SS2 infection, resulting in death. Survivors may subsequently suffer from SS2 crossing the blood-brain barrier (BBB) ​​and developing meningitis. Meningitis is the most severe manifestation of infection caused by Streptococcus suis type 2. Even after recovery, irreversible long-term sequelae such as sensorineural hearing loss may occur in humans. However, our understanding of the molecular mechanisms underlying meningitis induced by Streptococcus suis infection remains limited.

[0003] Studies have confirmed that virulence factors are important components of the infection mechanism of Streptococcus suis. To date, numerous virulence factors have been reported, some of which are considered crucial, such as GAPDH, Sly, Eno, and Cps. Increased Sly secretion promotes bacterial invasion of epithelial cells and enhances bacterial survival in the bloodstream, thereby promoting invasive infection of Streptococcus suis type 2. Eno binds to its receptor RPSA on the surface of brain microvascular endothelial cells, promoting HSPD1 expression via the p38-ERK-eIF4E pathway. HSPD1 translocates to the cytoplasm and interacts with ACTB, ultimately inducing apoptosis. Expression of the bimolecular signal transduction system VraSR helps SS2 resist PMN phagocytosis, promotes SS2 survival in peripheral blood, and facilitates infection. The cell wall anchoring protein SntA can directly activate the classical complement pathway and the lectin pathway, causing complement consumption and thus evading complement killing effects. Therefore, we propose the oahs gene deletion strain and its application in the prevention of Streptococcus suis type 2 infection. Summary of the Invention

[0004] The purpose of this invention is to provide an oahs gene deletion strain and its application in the prevention of Streptococcus suis type 2 infection, aiming to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The oahs gene deletion strain has the accession number CCTCC NO: M 20231488.

[0007] The method for preparing oahs gene deletion strains includes the following steps:

[0008] Upstream and downstream fragments of the oahs gene were amplified from the SC19 genome using primers Δoahs-F1 / Δoahs-R1 and Δoahs-F2 / Δoahs-R2.

[0009] The upstream and downstream fragments of the oahs gene were fused using overlap extension PCR and cloned into the thermosensitive suicide streptococcal shuttle vector pSET4s to generate the knockout vector pSET4s-Δoahs.

[0010] pSET4s-Δoahs was electroporated into SC19 strain using the Gene Pulser XCell electroporation system. Gene exchange strains were initially screened for resistance to spectinomycin at 100 ug / ml, and then the plasmid was discharged under temperature control.

[0011] After the recombinant plasmid pSET4s-Δoahs was electroporated into the SC19 strain, homologous recombination occurred between the recombinant plasmid and the bacterial genome, resulting in a strain with the oahs gene deletion.

[0012] Furthermore, the conditions for the step of electroporating pSET4s-Δoahs into SC19 plants are: voltage of 2500 V, capacitance of 25 μF, resistance of 200 Ω, and electroporation cup size of 1 mm.

[0013] Application of oahs gene deletion strain in the preparation of drugs for the prevention of Streptococcus suis type 2 infection.

[0014] Furthermore, the oahs gene-deleted strain after washing can be mixed with an adjuvant for use.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] 1. The oahs gene deletion strain, namely SC19-ΔOAHS, was identified as having very low virulence, essentially being avirulent. Immunization of mice with a high dose of this mutant strain caused virtually no infectious damage, and with the aid of adjuvants, antibody titers in mice significantly increased, successfully establishing targeted immune protection.

[0017] 2. Compared with ordinary live bacterial vaccines, the oahs gene-deleted strain is safer for immunization and is essentially harmless to mice. Moreover, after a normal immunization program, the established immune protection can help mice effectively resist infection by Streptococcus suis type 2, reducing mortality and bacterial colonization.

[0018] 3. The preparation method of the oahs gene deletion strain is simple, can be produced in a short time, and is easy to culture. As a vaccine, it requires no other complex processing; it can be used after washing and mixing with adjuvant. This oahs gene deletion strain has the potential to be used as a live bacterial vaccine against streptococcal infection in pigs. Attached Figure Description

[0019] Figure 1 The results of PCR using external primers for oahs gene identification in this invention are shown.

[0020] Figure 2 The results of PCR using primers for internal identification of the oahs gene in this invention are shown.

[0021] Figure 3 This invention uses Western blotting to detect the expression of OAHS protein in bacterial strains.

[0022] Figure 4 This is a graph showing the survival rate detection results of the present invention.

[0023] Figure 5 This is a graph showing the results of bacterial load testing in blood and various organs according to the present invention.

[0024] Figure 6 This is an observation of mouse organs 72 hours after immunizing mice with the oahs gene deletion strain according to the present invention.

[0025] Figure 7 This figure shows the evaluation results of the protective effect of the oahs gene deletion strain on mice immunized by this invention. In the figure, A: antibody titer detection; B: survival rate detection; C: post-infection weight change detection; D: bacterial load detection in blood and various organs. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0028] The oahs gene deletion strain provided in one embodiment of the present invention was deposited on August 17, 2023, at the China Center for Type Culture Collection (No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province), with accession number CCTCC NO: M 20231488, and classified as Streptococcus suis SC19-ΔOAHS.

[0029] In a preferred embodiment of the present invention, the gene sequence of the oahs gene deletion strain is SEQ ID No. 1.

[0030] As a preferred embodiment of the present invention, the type 2 Streptococcus suis strain SC19 used in the present invention was kindly provided by Professor Zhang Anding of Huazhong Agricultural University.

[0031] As a preferred embodiment of the present invention, the primers used to construct the strain are shown in Table 1.

[0032] Table 1 Primers for strain knockout

[0033]

[0034] An embodiment of the present invention provides a method for preparing an oahs gene deletion strain, comprising the following steps:

[0035] Using primers Δoahs-F1 / Δoahs-R1 and Δoahs-F2 / Δoahs-R2, the upstream (N-terminal) fragment (SEQ ID No. 2) and downstream (C-terminal) fragment (SEQ ID No. 3) of the oahs gene, each 1000 bp, were amplified from the SC19 genome.

[0036] The upstream and downstream fragments of the oahs gene were fused using overlap extension PCR and cloned into the thermosensitive suicide streptococcal shuttle vector pSET4s to generate the knockout vector pSET4s-Δoahs.

[0037] pSET4s-Δoahs was electroporated into SC19 strain using the Gene Pulser XCell electroporation system (Bio-Rad, Hercules, CA, USA) (voltage 2500 V, capacitance 25 μF, resistance 200 Ω, electroporation cup size 1 mm). Gene exchange strains were initially screened for resistance to spectinomycin at 100 ug / ml, and plasmids were then discharged under temperature control.

[0038] After the recombinant plasmid pSET4s-Δoahs was electroporated into the SC19 strain, homologous recombination occurred between the recombinant plasmid and the bacterial genome, resulting in a strain with the oahs gene deletion.

[0039] In this embodiment of the invention, a strain resistant to spectinomycin was selected and continuously passaged under antibiotic-free conditions to increase the frequency of further recombination. Primers Δoahs-F3 / R3 were designed 250 bp before and after the oahs gene on the genome, and primers Δoahs-F4 / R4 were designed inside the oahs gene. PCR was performed using these two primer pairs to verify the knockout effect. Specifically:

[0040] See Figure 1 Using primers Δoahs-F3 / R3, PCR amplification of the strain was performed. The oahs gene deletion strain only amplified a 500 bp band, while the negative control amplified a 1781 bp fragment (sequence SEQ ID No. 4). To further verify this, PCR amplification of the strain was performed using the internal primers Δoahs-F4 / R4 for the oahs gene sequence. The wild-type strain amplified a 660 bp band (sequence SEQ ID No. 5), while the mutant strain did not amplify this band. Figure 2 This further confirms the deletion of the target gene. Western blot experiments were performed on bacterial proteins using mouse serum immunized with OAHS protein as the primary antibody. The results also show that the knockout strain no longer expresses OAHS protein. Figure 3 In summary, the oahs gene deletion strain, namely SC19-ΔOAHS, was successfully constructed.

[0041] The application of the oahs gene deletion strain provided in one embodiment of the present invention in the preparation of a drug for preventing Streptococcus suis type 2 infection.

[0042] As a preferred embodiment of the present invention, the oahs gene deletion strain after washing is mixed with an adjuvant and can be used.

[0043] Example 1: SC19-ΔOAHS toxicity detection

[0044] Balb / c mice aged 6-8 weeks were randomly divided into a wild-type infection group and a mutant infection group (n=6). They were treated with SC19 and SC19-ΔOAHS at 2 times the LD50, respectively. 50 (approximately 1.1 × 10⁻⁶) 9 Mice were infected via intraperitoneal injection (CFU / mouse), and mortality was observed and recorded for seven consecutive days to plot the survival rate curve.

[0045] To detect bacterial load, 6-8 week old Balb / c mice were randomly divided into a wild-type infection group and a mutant infection group (n=3). SC19 and SC19-ΔOAHS were administered at 0.25 times the LD50, respectively. 50 (approximately 1.5 × 10) 8 Mice were infected intraperitoneally with CFU / mouse. 72 hours after infection, blood and internal organ tissues were collected. Blood was serially diluted and plated on BHI plates. Tissue was homogenized in PBS (3000 * tissue weight, unit: μL), serially diluted, and plated. After obvious bacterial growth, the colonies were counted and statistically analyzed.

[0046] Example 2: Immunization of mice with SC19-ΔOAHS live bacteria and its safety evaluation

[0047] Balb / c mice aged 6-8 weeks were immunized at 0, 14, and 28 days. SC19-ΔOAHS was mixed with aluminum gel adjuvant at a ratio of 7:1 (volume ratio: bacterial suspension:aluminum gel adjuvant = 7:1), with an initial immunization dose of 5 × 10⁻⁶. 8 CFU / animal, secondary immunization and booster immunization bacterial count is 2.5 × 10⁻⁶. 8 CFU / mouse. 200 μL / mouse was injected intraperitoneally. Control mice were simultaneously injected intraperitoneally with the same volume and proportion of adjuvant solution.

[0048] The mice were continuously observed and their condition recorded after the initial immunization. Seventy-two hours after the initial immunization, the mice were necropsed, and the bacterial load in their blood and organs was measured.

[0049] Results: All mice infected with the SC19 strain via intraperitoneal route died within 24 hours, while all mice infected with SC19-ΔOAHS survived after 7 days. Figure 4 ) 72 h after infection, the bacterial load in the blood and various tissues and organs of mice was measured. It was found that compared with mice infected with the SC19 strain, the bacterial load in all tissues and organs of mice infected with SC19-ΔOAHS was significantly reduced (except for the brain and kidney). Figure 5 ).

[0050] Six hours after the initial immunization, immunized mice showed mild signs of infection, but by 24 hours post-immunization, there was no significant difference compared to the control group, a pattern that persisted until day 7. At 72 hours post-immunization, the mice were necropsed, and bacterial load was measured. Results showed no bacterial colonization in the blood or organs of the immunized mice, and no ocular lesions were observed in the organs. Figure 6 These results indicate that immunization with SC19-ΔOAHS did not cause infectious damage in mice and is very safe as a live bacterial vaccine.

[0051] Example 3: Evaluation of the immunoprotective effect of SC19-ΔOAHS live bacterial vaccine

[0052] Seven days after booster immunization, immunized mice and control mice were infected with SC19, JZLQ022 (Streptococcus suis type 2), and JZLQ001 (Streptococcus suis type 7). Mouse body weight changes were measured within 72 hours post-infection. Bacterial load in blood and various organs was measured 72 hours post-infection. Mouse mortality was recorded up to 7 days post-infection, and survival rates were calculated and plotted as a line graph. Data from SC19-ΔOAHS immunized mice and control mice were compared to evaluate the protective efficacy of the mutant strain as a live bacterial vaccine.

[0053] Results: 35 days after immunization, the antiserum titer was detected by ELISA. The results showed that the OD450nm of the antiserum diluted 10¹ in immunized mice was significantly higher than that of the control group serum. Figure 7A). Mice were infected with SC19, JZLQ022, and JZLQ001 strains, respectively. Results showed that SC19-ΔOAHS immunization successfully protected mice against infection by SC19 and JZLQ022 strains, significantly increasing survival rate by 83.33%. However, immunization against different serotypes of JZLQ001 and SC19-ΔOAHS did not provide good protection, and there was no significant difference in survival rate between the immunized groups and the control group. Figure 7 B). Within 24 hours post-infection, the weight loss of immunized mice was generally not lower than that of unimmunized control mice, especially SC19-infected mice and JZLQ022-infected mice. The weight changes of immunized mice were significantly higher than those of control mice at 12 hours and 6 hours post-infection, respectively. Figure 7 C). At 72 h post-infection, the colonization level of SC19 in immunized mice was significantly lower than that in control mice (except for the spleen and kidneys). The colonization level of JZLQ022 was generally lower than that in non-immunized mice, although the difference was not significant. However, the colonization level of JZLQ001 was not significantly different from that in non-immunized mice. Figure 7 D).

[0054] In summary, SC19-ΔOAHS can successfully establish immunity in mice and has good immunoprotective effect against infection with the same type of Streptococcus suis, but its immunoprotective effect against infection with other serotypes of Streptococcus suis is generally limited.

[0055] Therefore, it can be seen that after the oahs gene deletion mutation, the highly virulent strain SC19 of Streptococcus suis type 2 becomes a non-virulent strain. Using its live bacteria, supplemented with adjuvant, to immunize mice has good safety and can effectively establish specific immune protection in mice, helping mice resist infection of its parent strain SC19 and other strains of the same serotype. It has the potential to be used as a live bacterial vaccine for swine against Streptococcus suis type 2 infection.

[0056] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A oahs gene deletion strain characterized in that, The preservation number of the oahs gene deletion strain is CCTCC NO: M20231488, and the classification name is Streptococcus suis SC19-ΔOAHS.

2. The oahs gene deletion strain of claim 1 in the preparation of a drug for preventing Streptococcus suis type 2 infection.

3. Use according to claim 2, characterized in that, The oahs gene deletion strain after washing bacteria is mixed with an adjuvant and can be used.

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