CN109820877B - Method for constructing treponema pallidum mouse model - Google Patents
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- CN109820877B CN109820877B CN201910293666.3A CN201910293666A CN109820877B CN 109820877 B CN109820877 B CN 109820877B CN 201910293666 A CN201910293666 A CN 201910293666A CN 109820877 B CN109820877 B CN 109820877B
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Abstract
The invention relates to the technical field of biology, and discloses a method for constructing a treponema pallidum mouse model. The method of the invention inoculates the treponema pallidum to a rabbit for breeding and culturing, and then inoculates the treponema pallidum in the skin, the peritoneum, the rectum and the cavernous body of the penis of a mouse to obtain the treponema pallidum mouse model. Aiming at the problems that the existing mice have very small reactivity to treponema pallidum infection and lack a treponema pallidum mouse model, the treponema pallidum is inoculated to the mice of specific strains in the skin, the peritoneum, the rectum and the cavernous penis simultaneously to construct the treponema pallidum mouse model, the mouse model can detect the treponema pallidum in blood, the liver, the spleen and the testis, can realize the blood and lymph node dissemination of the treponema pallidum, can reflect the inhibiting effect of the treponema pallidum DNA vaccine, and meets the requirements of serving as the treponema pallidum animal model.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for constructing a treponema pallidum mouse model.
Background
Syphilis is a chronic sexually transmitted disease caused by treponema pallidum. Currently, it is still a global epidemic with an estimated 3600 million cases worldwide. In recent years, the incidence of syphilis has increased dramatically in male sex-lovers. The incidence of congenital syphilis infection is also rising, and there are estimated to be 136 tens of thousands of pregnant women infected each year worldwide, of which about 520,000 cases lead to abortion or stillbirth, etc. In addition, syphilis increases the risk of HIV infection. Although treponema pallidum remains sensitive to penicillin treatment, global prevalence of syphilis still increases, and there is an urgent need to develop effective syphilis vaccines as a complement to traditional screening and treatment methods for global elimination of syphilis.
Development and research of the treponema pallidum vaccine require an animal model of the treponema pallidum to evaluate the immunogenicity and protective effect of a candidate vaccine on inhibiting the treponema pallidum infection. Mice have not been widely used in syphilis studies because of their low reactivity to treponema pallidum infection. The major animal model for syphilis studies is currently rabbits, but inbred rabbits are expensive and not readily available. The candidate vaccine in the existing research results can inhibit the spread of the treponema pallidum to a remote organ, but the research on the protective mechanism is difficult to enter the next step due to the lack of reagents for researching the rabbit humoral immunity and cellular immunity. Therefore, it is urgently needed to construct an animal model, and the immunoprotective agents required by the animal model are easy to obtain and the genetic manipulation of the immune system is common, so as to help researchers further study the immunogenicity and protectiveness of treponema pallidum flagellin for inhibiting the diffusion in the host body.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for constructing a treponema pallidum mouse model, so that the method can successfully construct a treponema pallidum infected mouse model, and can be used in the evaluation research of immunogenicity and protective effect of related vaccines for inhibiting treponema pallidum infection.
In order to achieve the above purpose, the invention provides the following technical scheme:
a method for constructing mouse model of treponema pallidum comprises inoculating treponema pallidum to rabbit, breeding, culturing, and inoculating treponema pallidum to mouse skin, intraperitoneal, rectal and cavernous penis to obtain mouse model of treponema pallidum.
Preferably, the treponema pallidum is inoculated into a rabbit for breeding and culturing, and the treponema pallidum is bred and cultured by inoculating an adult New Zealand white rabbit in a testis.
In a specific embodiment of the present invention, the number of treponema pallidum inoculated to the mouse intracutaneously, intraperitoneally, rectally and cavernosum penis is 2.5 × 106Treponema pallidum (total 1X 10)7One). Wherein the mouse skin is the skin between scapulae, and intrarectal inoculation adopts intragastric perfusion.
In a specific embodiment of the invention, the mouse is a C57BL/6 mouse. After C57BL/6 mouse is infected with syphilis for 30 days, treponema pallidum is detected in blood, liver, spleen, brain tissue and testis. Meanwhile, the mouse immunized by the DNA vaccine for inhibiting the treponema pallidum has obviously reduced carrying capacity of the treponema pallidum in each tissue.
Furthermore, lymph nodes of mice successfully infected with treponema pallidum were transfected into testis of normal New Zealand rabbits, and live treponema pallidum was found.
According to the test results, the invention considers that the blood vessel spreading of the treponema pallidum can be generated in the C57BL/6 mouse model as well as a human host, the requirement of the animal model for blood and lymph node spreading of the treponema pallidum is met, and the inhibition effect of the vaccine can be reflected. Therefore, the invention provides the application of the treponema pallidum mouse model constructed by the method in evaluating the immunogenicity and/or protective effect of the treponema pallidum vaccine.
Wherein, the treponema pallidum vaccine is preferably a treponema pallidum DNA vaccine.
According to the technical scheme, aiming at the problems that the existing mice have very low reactivity to treponema pallidum infection and lack of treponema pallidum mouse models, the treponema pallidum is inoculated in the skin, the peritoneum, the rectum and the cavernous penis of the mice of specific strains simultaneously to construct the treponema pallidum mouse model, the mouse model can detect the treponema pallidum in blood, liver, spleen and testis, can realize the blood and lymph node spreading of the treponema pallidum, can reflect the inhibition effect of the vaccine, and meets the requirements of serving as the treponema pallidum animal model.
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FIG. 1 shows the result of RT-qPCR detection of Treponema pallidum DNA in each tissue of mouse; wherein, A-F are the results of mouse blood, liver, spleen, lymph node, brain and testis in sequence; p < 0.05, p < 0.01, NS no significant difference;
FIG. 2 shows immunohistochemical results for mouse testis, liver and spleen tissues; wherein, A is testis, B is liver, and C is spleen.
Detailed Description
The invention discloses a method for constructing a treponema pallidum mouse model, which can be realized by appropriately improving process parameters by taking the contents of the text as reference by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations of parts, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
The method for constructing a treponema pallidum mouse model provided by the invention is further explained below.
Example 1: construction of Treponema pallidum mouse model and blood diffusion verification
Treponema pallidum is bred and cultured by inoculating adult New Zealand white rabbits in testis, inoculating Treponema pallidum in mouse skin (between shoulder blades), intraperitoneal, intrarectal (intragastric) and corpus cavernosum, each part is 2.5 × 106Treponema pallidum (total 1X 10)7One). Rectal inoculation adopts a gastric perfusion mode, and other parts adopt injection inoculation. Mice were evaluated every other day for signs of skin or systemic infection. Mice were sacrificed on day 30 post inoculation. Testis, lymph nodes (groin/brachium/axilla), spleen, liver, blood and brain tissue were collected for RT-qPCR detection analysis.
Mouse immunization: mice were randomly assigned, 9 per group. Mice are immunized by intramuscular injection, and both the pcDNA/CpG-FlaB3 and the pcDNA3/FlaB3 have the effect of inhibiting treponema pallidum. The first group was immunized with pcDNA/CpG-FlaB3 (100. mu.g); the second group was immunized with pcDNA3/FlaB3 (200. mu.g); as a blank control, mice were injected with pcDNA3 vector alone.
As shown in FIG. 1, the RT-PCR method is adopted to detect the load of the treponema pallidum in blood, lymph node, testis, brain, liver and spleen, each tissue of the control group shows higher load of the treponema pallidum, while the treatment group immunized by two DNA vaccines obviously reduces the load of each tissue, wherein the inhibition effect of pcDNA3/CpG-FlaB3 is higher.
Example 2: immunohistochemical verification of treponema pallidum mouse model
Mouse model construction and immunization same as example 1
Biopsy punch samples (2mm) were taken from testis, liver and spleen of each mouse 30 days post infection and sent to university of south China. Tissues were fixed in formalin and stained with hematoxylin and eosin or immunohistochemically stained with anti-T (figure 2).
The results in FIG. 2 show that a large number of treponema pallidum were found in testis, liver and spleen of the control group, whereas the spirochete load was significantly reduced in testis, liver and spleen of infected mice immunized with pcDNA3/FlaB3 and pcDNA3/CpG-FlaB 3.
Example 3: treponema pallidum mouse model lymph node spread verification
1) 30 days after Tp infection, C57BL/6 mice (3/group) were aseptically isolated for inguinal, brachial plexus, and axillary lymph nodes in sterile petri dishes, and 1mL of physiological saline was added;
2) fully grinding lymph nodes by using a medical injector (5mL) soft part, placing a culture dish on a platform rotator for 10min, removing lymph node fragments, and respectively sucking 2mL of bacterial liquid;
3) respectively infecting 3 New Zealand rabbits with Tp bacterial liquid collected from each group of mice, and injecting 1mL bacterial liquid after disinfecting testis with iodophor;
4) new Zealand rabbit testis inflammation was observed daily and serological tests (RPR and TPPA) were performed every other week;
5) after infection with 9w, new zealand rabbits were sacrificed under anesthesia, testis aseptically isolated, dark field microscopy and qRT-PCR to count Tp load in testis tissue.
The results are shown in Table 1
TABLE 1 serological response of New Zealand Rabbit lymph node transfection experiment
a'+' indicates that New Zealand rabbit serology TPPA and RPR reacted positively;btreponema pallidum load in the testis of new zealand rabbits.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (1)
1. A method for constructing treponema pallidum mouse model is characterized in that treponema pallidum is inoculated into an adult New Zealand rabbit through testis for breeding and culturing, and then treponema pallidum is inoculated into the skin, the peritoneum, the rectum and the penis cavernosum between the shoulder blades of a C57BL/6 mouse to obtain the treponema pallidum mouse model; the number of treponema pallidum inoculated in the skin, the peritoneum, the rectum and the cavernous penis among the shoulder blades of the mouse is 2.5 multiplied by 106Treponema pallidum; the intrarectal inoculation adopts intrarectal inoculation.
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