An etec vaccine and uses thereof

Abstract

The present application relates to the technical field of vaccines, and specifically discloses an ETEC vaccine and application thereof.The ETEC vaccine comprises Pichia pastoris.The ETEC vaccine is used for promoting animals to produce specific antibodies and increasing the expression amount of tight junction proteins and mucin.The application adds Pichia pastoris to the ETEC vaccine, which can significantly increase the specific antibodies in the animal body, improve the immune response of the body, increase the expression amount of intestinal tight junction proteins and mucin, protect the intestinal tract of the body, and further improve the immune protection effect of the ETEC vaccine, and has great application value.

Description

Technical Field

[0001] This invention relates to the field of vaccine technology, and more particularly to an ETEC vaccine and its application. Background Technology

[0002] Enterotoxigenic Escherichia coli (ETEC) is one of the pathogens causing diarrhea in young animals such as piglets, calves, and lambs, resulting in significant economic losses for the livestock industry. Due to the numerous serotypes of E. coli and their extremely complex antigenic composition, the prevalent serotypes vary considerably across regions, leading to unsatisfactory cross-protective effects between different serotypes. Currently, commercially available vaccines in China mainly consist of whole-cell bacteria containing K88, K99, and 987P. These vaccines not only contain components that can enhance the immunogenicity of animals but also contain components unrelated to immune protection and endotoxins, further contributing to poor vaccine efficacy. Therefore, improving the protective effect of inactivated ETEC vaccines is an urgent problem to be solved.

[0003] In recent years, many studies have found that administering vaccine adjuvants via gavage or injection can enhance the body's immune response and improve immune levels. However, commonly used vaccine adjuvants have certain toxicity, weak immunogenicity, and poor safety profiles. Therefore, researching a safe and effective immune adjuvant is of great significance for enhancing cellular immune responses and strengthening the immune response specific to vaccine component antigens. Summary of the Invention

[0004] In response to the problems of poor protective efficacy of existing ETEC vaccines, the toxicity and weak pathogenicity of vaccine adjuvants, and poor safety, this invention provides an ETEC vaccine that can significantly increase specific antibodies in animals, promote the expression of intestinal tight junction proteins and mucins, enhance intestinal barrier function, and improve the body's immune response to the ETEC vaccine.

[0005] Furthermore, the present invention also provides the use of the above-described ETEC vaccine in promoting the production of specific antibodies in animals.

[0006] Furthermore, the present invention also provides the application of the above-mentioned ETEC vaccine in increasing the expression levels of tight junction proteins and mucins.

[0007] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions:

[0008] An ETEC vaccine, comprising Pichia pastoris.

[0009] Compared to existing technologies, the ETEC vaccine provided in this application has the following advantages:

[0010] Young animals are prone to diarrhea after being injected with ETEC vaccine. The applicant accidentally discovered that adding Pichia pastoris liquid during ETEC vaccine injection can significantly increase specific antibodies in animals, enhance the body's immune response, improve the intestinal mucosa, promote the development and maturation of tissue structure, protect the intestines, and thus enhance the immune protection effect of ETEC vaccine.

[0011] This application adds Pichia pastoris to the ETEC vaccine, which can be used as a vaccine adjuvant for the inactivated ETEC vaccine. This solves the problems of certain toxicity, weak pathogenicity, and poor safety of vaccine adjuvants, and shows great application value and development potential in improving the protective effect of ETEC vaccine.

[0012] Pichia pastoris (P. pastoris) is a methyltrophic yeast with advantages such as high expression of exogenous genes, high genetic stability, and strong extracellular secretion ability. Its glucan and mannan have good effects on enhancing the body's immunity, improving blood lipid metabolism, and improving the body's antioxidant capacity. This may be the reason why Pichia pastoris can improve the vaccine response level and protect the body's intestines as a vaccine adjuvant.

[0013] Optionally, the Pichia pastoris is a live strain.

[0014] Optionally, the Pichia pastoris is an inactivated strain.

[0015] Both live and inactivated Pichia pastoris can enhance the immunoprotective effect and intestinal protective function of ETEC inactivated vaccine, with live Pichia pastoris showing better efficacy.

[0016] Optionally, the Pichia pastoris strain content is 0.8 × 10⁻⁶. 9 cfu / mL~1.2×10 9 cfu / mL.

[0017] Optionally, the strain number of the Pichia pastoris is GS115.

[0018] Furthermore, this application also provides the use of the aforementioned ETEC vaccine in promoting the production of specific antibodies in animals.

[0019] ETEC vaccines, including those based on Pichia pastoris, can significantly increase the level of specific antibodies in animals, effectively enhance the body's immune response to ETEC vaccines, and enrich the variety of adjuvants, providing new potential adjuvants for the prevention and treatment of E. coli diarrhea.

[0020] Furthermore, this application also provides the application of the aforementioned ETEC vaccine in increasing the expression levels of tight junction proteins and mucins.

[0021] ETEC vaccines, including those based on Pichia pastoris, can effectively improve the morphology of the intestinal mucosa in animals and promote the development and improvement of tissue structure; they can also promote the expression of intestinal tight junction protein mRNA and enhance the barrier function of the intestine.

[0022] Optionally, the specific antibody includes at least one of IgA or IgG.

[0023] Optionally, the tight junction protein includes at least one of Occludin or ZO-1.

[0024] Optionally, the mucin is MUC2. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This refers to the antibody level of mouse K88ac specific antibody IgA provided in the embodiments of the present invention;

[0027] Figure 2 This refers to the antibody level of mouse K88ac-specific antibody IgG provided in the embodiments of the present invention;

[0028] Figure 3 This refers to the mRNA expression level of the tight junction protein Occludin provided in the embodiments of the present invention;

[0029] Figure 4 This refers to the mRNA expression level of the tight junction protein ZO-1 provided in the embodiments of the present invention;

[0030] Figure 5 This refers to the mRNA expression level of the mucin MUC2 provided in the embodiments of the present invention;

[0031] Figure 6 This refers to the antibody level of K88ac-specific antibody IgA in mouse intestinal fluid provided in this embodiment of the invention.

[0032] Figure 7 These are mouse small intestine tissue slices provided in the embodiments of the present invention;

[0033] Figure 8 This refers to the mouse spleen index provided in the embodiments of the present invention;

[0034] Figure 9 This refers to the percentage of mouse body weight maintained after infection, as provided in the embodiments of the present invention.

[0035] Among them, Live Pichia+V: live Pichia + vaccine group; Inactivated Pichia+V: inactivated Pichia + vaccine group; Pichia RNA+V: Pichia RNA + vaccine group; Live SC+V: live Saccharomyces cerevisiae + vaccine group; V: vaccine alone group; Normal saline: saline group. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0037] Example 1

[0038] 1. Materials and Methods

[0039] 1.1 Bacterial strains and laboratory animals

[0040] ETEC K88ac was purchased from the China Veterinary Microbiology Culture Collection Center, with the strain number C1518.

[0041] Pichia pastoris strain, strain number GS115;

[0042] 6-8 week old SPF-grade BALB / c female mice were purchased from Beijing Spaford Biotechnology Co., Ltd.

[0043] 1.2 Main Reagents

[0044] Anhydrous ethanol, isopropanol, formaldehyde solution, and xylene were all analytical grade reagents purchased from Tianjin Jinfeng Chemical Co., Ltd. TRIzol was purchased from Invitrogen, USA. The mouse Escherichia coli K88ac antibody IgG (K88ac IgG) ELISA kit (catalog number: MM-45009M1) and mouse Escherichia coli K88ac antibody IgA (K88ac IgA) ELISA kit (catalog number: MM-45003M1) were purchased from Jiangsu Baolai (Enzyme Immunoassay) Biotechnology Co., Ltd.; the HiScript II 1st StrandcDNA Synthesis Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.; the IQTM SYBR Green Supermix kit was purchased from Bio-Rad Laboratories, USA; the trivalent inactivated vaccines for swine coliform infection K88, K99, and 987P were purchased from Shandong Huahong Biotechnology Co., Ltd.; yeast extract and tryptone were purchased from OXOID, UK; sodium chloride was purchased from Fuchen (Tianjin) Chemical Reagent Co., Ltd.; and all primers were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0045] 1.3 Preparation of yeast culture

[0046] The frozen Pichia pastoris culture stored at -80℃ was inoculated onto YPD solid medium and incubated at 30℃ for 4 days. Single colonies were picked using a sterile inoculation loop and added to liquid YPD medium, then incubated overnight at 225℃ on a shaker. 10 mL of the culture was then transferred to an Erlenmeyer flask, and 250 mL of liquid YPD medium was added. The culture was shaken until the absorbance at 600 nm measured with Nanodrop One was 2, indicating a viable culture. A portion of the viable culture was aliquoted into another Erlenmeyer flask and autoclaved at 121℃ for 20 minutes to obtain an inactivated culture. When the temperature dropped to 45℃, the culture was poured into plates near an alcohol lamp in a laminar flow hood and allowed to solidify before use. The viable and inactivated cultures were centrifuged to collect the cells. The supernatant was removed, and the precipitate was washed with physiological saline, centrifuged again, and resuspended in sterile physiological saline to prepare a suspension. The resulting Pichia pastoris cultures had a cell count of 1×10⁻⁶. 9 Live and inactivated bacterial solutions at CFU / mL.

[0047] The preparation process of the above solid YPD medium is as follows: 2g of agar is added to 100mL of YPD liquid medium to prepare a liquid. After being autoclaved at 121℃ for 20min, the liquid is cooled to 45℃, poured into a plate and inverted to solidify, which is the solid YPD medium.

[0048] The live brewer's yeast solution was also obtained using the above method, and its bacterial count was also 1×10⁻⁶. 9 cfu / mL.

[0049] 1.4 Extraction of Pichia pastoris RNA

[0050] Pichia pastoris live bacterial suspension was prepared using the procedure described in 1.3. The cells were collected by centrifugation, and the supernatant was discarded. The mortar and pestle were pre-cooled with liquid nitrogen, and the yeast cells were ground into a fine powder using liquid nitrogen. 80 mg of the powder was added to an RNase-free centrifuge tube, and 1 mL of Trizol was immediately added. The mixture was vigorously vortexed and centrifuged at room temperature to collect the supernatant. The supernatant was transferred to a new centrifuge tube, and 0.2 mL of chloroform was added. After vortexing, the tube was placed on ice, and the supernatant was collected by centrifugation. An equal volume of isopropanol was added to the supernatant, and the mixture was inverted to mix. The tube was then incubated at -20°C, and the supernatant was removed by centrifugation. The precipitate was washed with 1 mL of 75% DEPC water-ethanol solution, and then the supernatant was discarded by centrifugation. The tube was left unsealed in a clean bench for several minutes to allow the ethanol to evaporate completely. An appropriate amount of DEPC water was added to the tube to fully dissolve the RNA to a concentration of 1000 ng / μL–2000 ng / μL.

[0051] 1.5 Immunization and challenge in mice

[0052] Seventy-two SPF-grade BALB / c female mice were divided into six groups of 12 mice each: a live Pichia pastoris vaccine group, an inactivated Pichia pastoris vaccine group, a Pichia pastoris nucleic acid vaccine group, a live Saccharomyces cerevisiae vaccine group, a single vaccine group, and a saline group. After a 7-day acclimatization period, the mice were immunized. Each mouse received an intramuscular injection of a mixture of bacterial suspension and ETEC vaccine, with 10 μL of inactivated ETEC vaccine and 90 μL of either live or inactivated bacterial suspension. In the Pichia pastoris nucleic acid vaccine group, the vaccine dose was 10 μL, the RNA dose was 100 μg, and the remaining volume was made up with saline. Fifteen days post-immunization, six mice from each group were collected for sample collection and analysis of various indicators. Simultaneously, the remaining mice in each group were challenged with an intraperitoneal injection of 0.3 mL of ETEC K88ac. The number of surviving mice and changes in body weight were observed for five consecutive days.

[0053] 1.6 Sample Collection

[0054] On day 15 post-immunization, six mice from each group were collected, their weight was recorded, blood was collected, serum was separated by centrifugation and aliquoted and frozen at -80°C for later use. Small intestinal tissue was then collected and flash-frozen in liquid nitrogen, and then transferred to -80°C for storage, followed by RNA extraction. A segment of small intestinal tissue was soaked in 10% formaldehyde solution and then stained with hematoxylin and eosin (HE). In addition, the weight of the mouse spleen was recorded.

[0055] 1.7 Determination of serum immunoglobulins

[0056] Serum samples were collected and processed according to the procedures of an enzyme-linked immunosorbent assay (ELISA) kit to determine the concentrations of IgA and IgG in the serum. The results were analyzed using SPSS for significance analysis, employing the independent samples criterion. P > 0.05 indicated no significant difference between groups, P < 0.05 indicated a significant difference between groups, and P < 0.01 indicated an extremely significant difference between groups.

[0057] 1.8 RNA extraction from small intestinal tissue

[0058] Weigh 100 mg of mouse small intestine tissue, add 1 ml of TRIzol and homogenize in a glass homogenizer. Collect the homogenate into a centrifuge tube, vortex vigorously, and centrifuge to collect the supernatant. Add 200 μL of chloroform to the supernatant, vortex and let stand, centrifuge and transfer the supernatant to another RNase-free centrifuge tube. Add an equal volume of isopropanol to the supernatant, invert and mix well, and let stand at -20°C. Then centrifuge and discard the supernatant. Add 75% ethanol prepared with DEPC water, gently tap the tube wall to suspend the precipitate, let stand, centrifuge and discard the supernatant, and air dry at room temperature until the precipitate is transparent. Add an appropriate amount of DEPC water to dissolve the precipitate, measure the RNA concentration using Nanodrop One and dilute to 1000 ng / μl-2000 ng / μl, and store at -80°C for later use.

[0059] 1.9 Quantitative Real-Time PCR (RT-qPCR)

[0060] RT-qPCR primers were designed based on mRNA sequences from NCBI. RNA from small intestinal tissue was reverse transcribed according to the HiScript II 1st Strand cDNA Synthesis Kit. Amplification was performed using SYBR Green, with the following program: Preincubation 95℃ / 600s; 3-Step Amplification 95℃ / 10s, 60℃ / 10s, 72℃ / 10s, 40 cycles; Melting 95℃ / 10s, 65℃ / 60s, 97℃ / 1s; Cooling 37℃ / 30s. β-actin was used as an internal reference gene. The relative transcription levels of each gene were calculated using the 2-ΔΔCt method. Brief descriptions of the target genes and primers are shown in Table 1.

[0061] Table 1 Primer sequences used for RT-qPCR

[0062]

[0063] 1.10 Paraffin sections and HE staining of small intestine tissue

[0064] Soak small intestine tissue in a 10% formaldehyde solution for at least two weeks until the tissue turns white, then trim the tissue blocks to 2mm. 3 ~3mm 3First, the small intestine tissue was incubated overnight in 70% ethanol, then dehydrated using different gradients of ethanol solutions (volume concentration): 80% for 2 hours; 85% for 1 hour; 90% for 1 hour; 95% for 1 hour; 100% I for 1 hour; 100% II for 30 minutes; and ethanol / xylene (volume ratio 1:1) for 5 minutes. After clearing in xylene for 2 minutes, it was then immersed in wax I at 65°C for 1 hour; and in wax II at 70°C for 30 minutes. The wax-infused small intestine tissue was vertically embedded in the bottom center of the wax box and placed on a cryostat overnight. Sections were cut to 5 mm in diameter, spread in 45°C warm water, transferred to glass slides, labeled, and dried in an oven at 50°C–60°C. After drying, the tissue was stained with hematoxylin and eosin (HE), mounted with resin, and dried before microscopic observation of the structural integrity of the small intestine tissue.

[0065] 2 Results

[0066] 2.1 Mouse K88ac-specific antibody levels

[0067] The antibody levels of specific antibodies IgA and IgG against the ETEC K88ac vaccine in mice are as follows: Figure 1 and Figure 2 As shown. Figure 1 and Figure 2 In the middle, the horizontal axis represents: Live Pichia+V: Pichia pastoris live bacteria + vaccine group; Inactivated Pichia+V: Pichia pastoris inactivated bacteria + vaccine group; Pichia RNA+V: Pichia pastoris nucleic acid + vaccine group; Live SC+V: Saccharomyces cerevisiae live bacteria + vaccine group; V: vaccine alone group; Normal saline: saline group. Figure 1 and Figure 2 The vertical axis represents the antibody levels of K88ac specific antibodies IgA and IgG in the serum of mice in each group.

[0068] from Figure 1 As can be seen, the K88ac IgA level in the Pichia pastoris live vaccine group was significantly higher than that in other groups; the Pichia pastoris inactivated vaccine group and the Saccharomyces cerevisiae live vaccine group were significantly higher than those in the single vaccine group and the saline group; and the Pichia pastoris inactivated vaccine group was significantly higher than that in the Pichia pastoris nucleic acid vaccine group.

[0069] from Figure 2 As can be seen, the levels of K88ac IgG in the Pichia pastoris live vaccine group and the Pichia pastoris inactivated vaccine group were significantly higher than those in the other four groups, and there was a significant difference between the Pichia pastoris live vaccine group and the Pichia pastoris inactivated vaccine group. Compared with the saline group, the addition of Pichia pastoris nucleic acid and Saccharomyces cerevisiae live cells can significantly increase the level of K88ac IgG, and the level of K88ac IgG in the Saccharomyces cerevisiae live vaccine group is significantly higher than that in the vaccine group.

[0070] from Figure 1 and Figure 2 It can be seen that both live and inactivated Pichia pastoris cells can produce more specific antibodies, thus more effectively improving the immunoprotective effect of inactivated vaccines.

[0071] 2.2 Intestinal barrier function in mice

[0072] The results of expression levels of tight junction proteins and mucins in mice are as follows: Figures 3-5 As shown. Figures 3-5 In the middle, the horizontal axis represents: Live Pichia+V: Pichia pastoris live bacteria + vaccine group; Inactivated Pichia+V: Pichia pastoris inactivated bacteria + vaccine group; Pichia RNA+V: Pichia pastoris nucleic acid + vaccine group; Live SC+V: Saccharomyces cerevisiae live bacteria + vaccine group; V: vaccine alone group; Normal saline: saline group. Figures 3-5 The vertical axis represents the mRNA expression levels of the tight junction proteins Occludin, ZO-1, and mucin MUC2 in the small intestine of each group of mice.

[0073] from Figures 3-5 The results show that both live and inactivated Pichia pastoris significantly increased the expression levels of tight junction proteins and mucins. The expression levels of Occludin and ZO-1 in the live Pichia pastoris vaccine group were significantly higher than those in the inactivated Pichia pastoris vaccine group, the Pichia pastoris nucleic acid vaccine group, the Saccharomyces cerevisiae live vaccine group, and the single vaccine group. The Occludin expression level in the Pichia pastoris nucleic acid vaccine group was significantly higher than that in the saline group; there were no significant differences among the Saccharomyces cerevisiae live vaccine group, the single vaccine group, and the saline group. Therefore, both live and inactivated Pichia pastoris can increase the expression levels of tight junction proteins and mucins in mice, and both have a protective effect on the intestines. However, live Pichia pastoris can better enhance the protective effect of the inactivated vaccine.

[0074] The antibody levels of K88ac-specific IgA in mouse intestinal fluid are as follows: Figure 6 As shown, from Figure 6The results show that the K88ac IgA antibody content in the Pichia pastoris live vaccine was significantly higher than that in the other five groups. Compared with the saline group, the K88ac IgA antibody content was significantly increased after the addition of live Pichia pastoris, inactivated Pichia pastoris, Pichia pastoris nucleic acid, and live Saccharomyces cerevisiae. The intestinal fluid K88ac IgA content in the live Saccharomyces cerevisiae group was significantly higher than that in the vaccine-only group. Therefore, both live and inactivated Pichia pastoris can increase the level of K88ac IgA antibodies in intestinal fluid and have a protective effect on the intestines. However, the effect of live Pichia pastoris is more pronounced, better enhancing the protective effect of the inactivated vaccine and superior to that of live Saccharomyces cerevisiae.

[0075] 2.3 Effects of Pichia pastoris on intestinal structure and integrity in small intestine tissue

[0076] Table 2 shows the results of villus height (VH), crypt depth (CD), and villus-to-crypt ratio (V / C) of the small intestinal tissue. As can be seen from Table 2, the villus height and villus-to-crypt ratio of live Pichia pastoris were significantly higher than those of the other groups, while the crypt depth was lower. This indicates that both live and inactivated Pichia pastoris can increase the height of the small intestinal villi and provide some protection to the intestinal mucosa, with live Pichia pastoris exhibiting stronger protective effects.

[0077] In Table 2: Live Pichia+V: Pichia pastoris live bacteria + vaccine group; Inactivated Pichia+V: Pichia pastoris inactivated bacteria + vaccine group; Pichia RNA+V: Pichia pastoris nucleic acid + vaccine group; Live SC+V: Saccharomyces cerevisiae live bacteria + vaccine group; V: vaccine alone group; Normal saline: saline group.

[0078] Table 2. Small intestinal villus height, crypt depth, and villus-crypt ratio

[0079] VH CD V / C Live Pichia+V <![CDATA[716.25±43.47 A ]]> <![CDATA[121.30±13.57 Cc ]]> <![CDATA[5.96±0.63 A ]]> Inactivated Pichia+V <![CDATA[561.77±19.96 Ba ]]> <![CDATA[132.85±15.49 Bc ]]> <![CDATA[4.30±0.66 B ]]> Pichia RNA+V <![CDATA[553.87±58.90 B ]]> <![CDATA[147.83±25.15 ab ]]> <![CDATA[3.82±0.64 BaCD ]]> Live SC+V <![CDATA[588.33±25.15 B ]]> <![CDATA[174.60±21.52 A ]]> <![CDATA[3.12±0.45 CbD ]]> V <![CDATA[531.12±39.17 Bb ]]> <![CDATA[137.92±17.84 Bbc ]]> <![CDATA[3.91±0.56 BCc ]]> Normal saline <![CDATA[455.08±25.14 C ]]> <![CDATA[139.07±13.01 B ]]> <![CDATA[3.30±0.36 Dd ]]>

[0080] Note: Different lowercase letters in the same column indicate significant differences (P<0.05), different uppercase letters indicate extremely significant differences (P<0.01), and the same letter or no letter indicates no significant differences (P>0.05).

[0081] Results of mouse small intestine tissue sections Figure 7 As shown, from Figure 7 It can be seen that using Pichia pastoris as a vaccine adjuvant can increase the height of the small intestinal villi, thereby protecting the integrity of the intestine.

[0082] 2.4 Spleen index in mice

[0083] The spleen index results of mice in each group are as follows: Figure 8 As shown. Figure 8 In the graph, the horizontal axis represents: Live Pichia+V: Live Pichia + Vaccine group; Inactivated Pichia+V: Inactivated Pichia + Vaccine group; Pichia RNA+V: Pichia RNA + Vaccine group; Live SC+V: Live Saccharomyces cerevisiae + Vaccine group; V: Vaccine alone group; Normal saline group; and the vertical axis represents the spleen index of each group of mice. The spleen index, which is the ratio of the mouse's spleen weight to its body weight, reflects the body's immune level. The spleen is an important peripheral immune organ and a site for immune cell residence; therefore, the spleen index can also reflect the level of immunity.

[0084] from Figure 8 The results show that, compared with the vaccine alone and the saline group, the addition of live Pichia pastoris and inactivated Pichia pastoris vaccines significantly or substantially increased the spleen index; the spleen index of the live Pichia pastoris vaccine was significantly higher than that of the live Saccharomyces cerevisiae vaccine. Therefore, both live and inactivated Pichia pastoris can enhance the immunity and spleen index of mice, with live yeast showing a more significant effect on improving the immunity of mice than inactivated yeast.

[0085] 2.5 Changes in body weight in mice infected with ETEC K88ac

[0086] The weight changes of mice in each group were recorded for five consecutive days as follows: Figure 9 As shown, from Figure 9 As can be seen, all groups experienced a decrease in body weight on day 1 post-infection, with the saline group showing the most significant decrease. The Pichia pastoris live vaccine group, the Pichia pastoris inactivated vaccine group, and the Pichia pastoris nucleic acid vaccine group all experienced a increase in body weight on day 2 post-infection, with the live Pichia pastoris vaccine group having a higher body weight than the other two groups. The Saccharomyces cerevisiae live vaccine group, the single vaccine group, and the saline group all showed a decrease in body weight on day 2. Body weight gradually increased from day 3 post-infection. On day 5, there was no significant difference in body weight among the live Pichia pastoris vaccine group, the inactivated Pichia pastoris vaccine group, and the Pichia pastoris nucleic acid vaccine group. Therefore, live and inactivated Pichia pastoris can maintain the body weight of mice infected with ETEC K88ac.

[0087] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An ETEC vaccine, characterized in that: Composed of Pichia pastoris RNA and trivalent inactivated vaccine; The strain number of Pichia pastoris is GS115; The trivalent inactivated vaccine is the K88, K99, and 987P trivalent inactivated vaccine for swine coliform infection.

2. The use of the ETEC vaccine as described in claim 1 in the preparation of a medicament for protecting the gut.

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