CN103352047B - Trichinella Spiralis larva ES antigen gene vaccine and preparation method - Google Patents

Abstract

The invention discloses a Trichinella Spiralis larva ES antigen gene vaccine and a preparation method thereof. Trichinella Spiralis larva 43ku and 45ku ES protein genes are respectively inserted to a pVAXI eukaryotic expression vector to obtain recombinant plasmids, and the recombinant plasmids are mixed according to a ratio of 1:1 to prepare a combined recombined DNA vaccine for preventing Trichinella Spiralis infection and improving the worm reduction rate of bodies. After the injection immunization of hindlimb muscles of mice for three weeks, 2000 worms are killed, the worm reduction rate can reach 76.65%, and the quantity of eggs at the tongue tissues of the mice is obviously reduced.

Description

Trichinella spiralis muscle larva ES antigen gene vaccine and preparation method thereof

technical field

The invention belongs to the field of biological technology, and relates to a trichinella spiralis muscle larva ES antigen gene vaccine and a preparation method thereof.

Background technique

Trichinella spiralis parasitizes humans and various vertebrates, and is a zoonotic parasitic disease that causes trichinellosis. Trichinella can parasitize humans and more than 150 kinds of animals. The main cause of infection is the intake of raw or undercooked meat products with cysts of Trichinella muscle larvae. resulting in the death of the patient. It is estimated that there are approximately 11 million infected people in the world at present. After 1975 in my country, there were also reports of outbreaks of this disease in Jilin, Liaoning, Heilongjiang, Henan, Hubei and other provinces and municipalities. The disease not only seriously endangers human health, but also causes huge economic losses to the pig industry and meat export. The International Veterinary Bureau reported that 10,000 live pigs were infected in 1998. There are pig trichinellosis in 26 provinces and municipalities in my country. It is prevalent. In the market trade markets in the vast rural areas of our country, pork or pork products that have not been quarantined by Trichinella spiralis are often sold without permission after slaughtering, which has brought great potential threats to human health and animal health. However, there is currently no effective vaccine against Trichinella spiralis.

The developmental stages of Trichinella spiralis include adult stage, newborn larvae stage and muscle larvae stage. The body antigens are diverse, mainly including surface antigens, body antigens, excretion-secretion antigens (ES antigens), and rod cell granule-associated antigens. Studies have shown that Trichinella spiralis ES antigen can be produced in three stages, which is directly exposed to the host's immune system and is the most important protective antigen for hosts against Trichinella spiralis infection, and is also the most important spiralis in current research. caterpillar antigen.

Trichinella spiralis ES antigen is the antigen substance secreted or excreted by its natural pores, which can be produced by the parasites of different stages, and the main secretion site is the rod-shaped cells near the esophageal tube of the parasite. ES antigens are composed of various protein components. Despommier et al. used SDS-PAGE to find that there were 28 antigenic components in the ES antigen of Trichinella spiralis. Using IEF method, 37 kinds of antigenic components could be found, including 22 kinds of glycoproteins; domestic scholars such as Zhu Xingquan also The composition of the ES antigen of Trichinella spiralis muscle larvae is very complex. Studies have found that 43ku and 45ku ES antigens can induce the body to produce major protective antibodies.

Trichinella spiralis 43ku antigen is the main protein component of ES antigen, which is highly conserved in Trichinella spiralis. Studies have shown that 43ku ES antigen is related to host nutrition, cell formation, excretion response and protective immune response, and 43ku ES antigen glycoprotein has been used as a good candidate antigen for immunodiagnosis and vaccine development.

The 45 ku antigen of Trichinella spiralis is a secreted glycoprotein, and it is also one of the main antigenic components of ES products of Trichinella spiralis muscle larvae. Arasu et al. first cloned the 45 ku antigen gene, and its recombinant protein could be recognized by the serum of Trichinella-infected mice. Trichinella spiralis 45 ku antigen is presumed to be a multigene family of serine proteases, encoding a protein of muscle larvae, but the active center of the enzyme is removed. The study also found that the antigen not only has good immunogenicity, but also has strong immune protection against Trichinella spiralis infection. The latest research shows that this gene is expressed in Trichinella spiralis muscle larvae and adults, and the 45 ku antigen of Trichinella spiralis includes peptide epitopes and sugar epitopes. The species-specific epitope of Trichinella spiralis can be recognized by monoclonal antibody. Wang Zhongquan et al. used RT-PCR method to clone and express the 45 ku antigen gene of Trichinella spiralis. Among the amplified 890 bases, there are genes reported by American scholars. The sequence homology reached 99.6%, which indicated that the 45ku antigenic protein gene was highly conserved, and it was a good candidate gene for DNA vaccine development.

DNA vaccine refers to the direct injection of a recombinant eukaryotic expression vector encoding a certain antigen into the animal body, so that the foreign gene is expressed in vivo, and the generated antigen activates the immune system of the body, thereby inducing specific humoral immunity and cellular immune response . The vaccine not only has the advantages of attenuated vaccines, but also has no risk of reversal, so it has attracted more and more attention, and is regarded as the third generation of vaccines after traditional vaccines and genetically engineered subunit vaccines.

Contents of the invention

The object of the present invention is to provide a combined recombinant DNA vaccine which effectively prevents Trichinella spiralis infection and improves the worm reduction rate and its preparation method.

The eukaryotic expression vector pVAXI -43ku of the recombinant 43ku ES antigen gene of Trichinella spiralis muscle larvae, which is inserted into the eukaryotic expression vector pVAXI with the 43ku gene, the base sequence of the 43ku is shown in the sequence table SEQID No.1;

The eukaryotic expression vector pVAXⅠ -45ku of the recombinant 45ku ES antigen gene of Trichinella spiralis muscle larvae, which is inserted into the eukaryotic expression vector pVAXⅠ with the 45ku gene, the base sequence of the 45ku is shown in the sequence table SEQID No.2;

Trichinella spiralis muscle larvae ES antigen gene vaccine, which includes: recombinant plasmids pVAXI- 43ku and pVAXI- 45ku ;

Their weight ratio is 1:1;

The concentrations of both pVAXI- 43ku and pVAXI- 45ku were 1 μg/μL.

Trichinella spiralis muscle larvae ES antigen gene vaccine preparation method, comprising:

(1) Prepare the total RNA of Trichinella spiralis muscle larvae, reverse transcribe it into cDNA, and use the cDNA as a template with primers:

43ku-P1:5'-CGCGGTACCATGCGAATATACATTTTTCTTAG-3'

43ku-P2: 5'-CGAGGATCCTTAGCTGTATGGGCAA-3'

45ku-P1: 5'-CGCGGTACCATGAAACTCTTGCTTTTAACA-3'

45ku-P2: 5'-GCGGATCCTTAGCCTTGCTTAGAGAG-3'

The PCR method amplifies the 43ku and 45ku ES antigen genes respectively, and the base sequences of the 43ku and 45ku genes are shown in the sequence table SEQID No.1 and 2;

(2) Insert the 43ku and 45ku genes into pVAXI eukaryotic expression vectors respectively to construct recombinant eukaryotic expression vectors pVAXI- 43ku and pVAXI- 43ku ;

(3) The two recombinant eukaryotic expression vectors were transformed into E.coli DH5α respectively, inoculated in LB liquid medium, and cultured on a shaker at 37°C and 150-180 r/min.

(4) Prepare a large amount of pVAXI- 43ku and pVAXI- 45ku from the above-mentioned cultured E.coli DH5α, adjust the concentration of the two, and mix them.

The invention provides a Trichinella spiralis muscle larva ES antigen gene vaccine and a preparation method thereof. The antigenicity of the Trichinella spiralis 43ku and 45ku ES proteins is used to prepare a combined recombinant DNA vaccine that can be used to prevent Trichinella spiralis infection and improve the rate of body reduction. After three weeks of immunization by intramuscular injection of the hind limbs of mice, 2000 worms were challenged, and the worm reduction rate could reach 76.65%, and the number of worm eggs in the mouse tongue tissue was significantly reduced.

The advantages of the present invention are: (1) The selected 43ku and 45ku ES proteins are protective antigenic proteins in Trichinella spiralis in each stage, which is beneficial for the body to remove the worms in different stages; (2) The vaccine type belongs to DNA vaccine with simple structure, The purification process is simple, the production cost is low, and it is suitable for mass production; (3) DNA vaccine molecules are stable, and DNA vaccines can be made into freeze-dried vaccines, and the original activity can be restored in saline solution when used, which is convenient for transportation and storage; ( 4) DNA vaccines are safe, eliminating the danger of easy reversal and anti-virus of traditional vaccines.

Description of drawings

Figure 1. PCR amplification of 43ku and 45ku antigen genes; among them, A: 43ku antigen gene PCR amplification; B: 45ku antigen gene PCR amplification

Figure 2. Identification of pMD18T- 43ku and pMD18T- 45ku enzyme digestion; Among them, A: identification of pMD18T- 43ku enzyme digestion; 2-B: identification of pMD18T- 45ku enzyme digestion;

Fig. 3. Preparation of eukaryotic expression plasmid vector pVAXⅠ;

Figure 4. Enzyme digestion and identification of recombinant expression vectors pVAXⅠ- 43ku and pVAXⅠ- 45ku ; among them, A: pVAXⅠ- 43ku restriction enzyme digestion; B: pVAXⅠ- 45ku restriction digestion;

Figure 5. Indirect immunofluorescence detection of MA104 cells transfected with pVAXⅠ- 43ku and pVAXⅠ- 45ku ; Among them, A: pVAXⅠ- 43ku transfected; B: pVAXⅠ- 45ku transfected; C: pVAXⅠ-transfected (control); D pVAXⅠ- 43ku immunized mice to challenge the worms;

Figure 6. The results of histopathological examination of mouse tongue; where, A: Normal mice were not challenged with worms; B PBS-administered mice were challenged with worms; C pVAXⅠ-immunized mice were challenged with worms; E: pVAXⅠ- 45ku immunized mice were challenged worms; F co-immunized mice challenged worms.

Detailed ways:

Example 1 Acquisition of 43ku and 45ku antigen genes of Trichinella spiralis muscle larvae

(1) Primer design and synthesis

According to the gene sequences of 43ku and 45ku ES antigens of Trichinella spiralis muscle larva registered in GenBank, Primers 5.0 was used to design primers, and Kpn Ⅰ and BamH Ⅰ restriction sites were designed on the upstream and downstream primers of the target gene, respectively. The primer sequences were provided by Shanghai Sangong Technology Co., Ltd. Synthesized by Bioengineering Ltd. The primer sequence is as follows

43ku upstream primer (43-P1): 5'-CGC GGTACC ATGCGAATATACATTTTTCTTAG-3'

43ku downstream primer (43-P2): 5'-CGA GGATCC TTAGCTGTATGGGCAA-3'

45ku upstream primer (45-P1): 5'-CGC GGTACC ATGAAACTCTTGCTTTTAACA-3'

45ku downstream primer (45-P2): 5'-GC GGATCC TTAGCCTTGCTTAGAGAG-3'

(2) PCR amplification of 43ku and 45ku antigen genes

Trichinella spiralis muscle larvae were prepared by the conventional Trizol method (total RNA, reverse-transcribed into cDNA, and cDNA was used as a template, and the 43ku and 45ku antigen genes were respectively amplified by PCR using the above primers. Trichinella spiralis is an international standard strain, T.spiralis (strain ISS 534), kept by our laboratory.

PCR operating conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 57°C for 1 min, extension at 72°C for 1 min, a total of 35 cycles; total extension at 72°C for 10 min. After the reaction, 3 μl of the product was taken for identification by 0.8% agarose gel electrophoresis, and the results showed that the 43ku and 45ku antigen genes were successfully amplified, as shown in FIG. 1 .

(3) Connection and transformation of 43ku and 45ku antigen genes with pMD18T

The DNA gel recovery kit from Axygen Company was used to recover the amplified target gene according to the instructions, and the recovered fragments were ligated with the cloning vector pMD18T.

Ligation conditions: overnight at 16°C.

The ligation product was transformed into E.coli DH5α competent cells by conventional CaCl ₂ method, and recombinant positive clones were screened.

(4) Identification and sequence analysis of pMD18T- 43ku and pMD18T- 45ku

Extract and screen suspected positive clone plasmids according to the instructions of the plasmid mini-extraction kit. The extracted plasmid vectors were digested and identified with Kpn Ⅰ and BamH Ⅰ restriction endonucleases. The restriction enzyme digestion system is as follows:

Reaction conditions: 37°C water bath for 2 h. After the end, the 0.8% agarose gel electrophoresis test showed that the recombinant expression vectors pMD18T- 43ku and pMD18T- 45ku were successfully obtained, as shown in Figure 2.

The recombinant plasmids pMD18T- 43ku and pMD18T- 45ku were sent to Beijing Huada Gene Technology Co., Ltd. for sequencing. Blast analysis of the sequencing results showed that the sequence similarity between the amplified 43ku ES antigen gene and the GenBank entry was 100%. , 45ku ES antigen gene was 97%.

Example 2 Preparation of eukaryotic recombinant expression vectors pVAXI- 43ku and pVAXI- 45ku

The eukaryotic expression vector pVAXI was purchased from Invitrogen, USA, and pVAXI was prepared according to the instructions of the plasmid mini-extraction kit, as shown in Figure 3.

(1) Recovery, ligation and transformation of the target gene

Use Kpn Ⅰ and restriction endonuclease to perform double digestion on pMD8T- 43ku , pVAXⅠ-45ku and pVAXⅠ, respectively, to obtain 43ku, 45ku ES antigen gene and pVAXⅠ large fragment; the double digestion system is as follows:

 

Reaction conditions: 37°C water bath for 3 h. After the end, follow the instructions of the DNA gel recovery kit to recover the target gene fragment.

The 43ku and 45ku ES antigen genes were respectively connected to the large fragment of the pVAXⅠ vector. The connection system is as follows:

Reaction conditions: connect at 21°C for 4 h, and overnight at 4°C; the next day, conventional CaCl ₂ method was used to transform E.coli DH5α competent, and screen for recombinant positive clones.

(2) Enzyme digestion identification of recombinant expression vectors pVAXⅠ- 43ku and pVAXⅠ- 45ku

The extracted plasmid vectors were digested and identified with Kpn Ⅰ and BamH Ⅰ restriction endonucleases. The restriction enzyme digestion system is as follows:

Reaction conditions: 37°C water bath for 2 h. After the end, the 0.8% agarose gel electrophoresis test showed that the recombinant expression vectors pVAXI- 43ku and pVAXI- 45ku were successfully constructed, as shown in Figure 4. The correctly identified recombinant E.coli DH5α was inoculated in LB liquid medium, cultured on a shaker at 37°C, 150-180 r/min, and a large number of recombinant eukaryotic expression vectors were prepared using a plasmid extraction kit.

Example 3 Transfection of pVAXⅠ- 43ku and pVAXⅠ- 45ku into MA104 cells and identification of expressed products

According to the instructions of Lipofectamine ^TM 2000, pVAXI- 43ku and pVAXI- 45ku were transfected into MA-104 cells respectively, and pVAXI was used as blank control.

After the transfection, the MA-104 cells were covered with coverslips and taken out, dried and washed once with PBS (pH7.2), fixed with 10% acetone for 10-15 min, washed three times with PBS (pH7.2), After blotted the edge moisture, add mouse-derived 43ku and 45ku ES antigen monoclonal antibodies respectively, place in a 37°C incubator for 2 hours, take out the cover slip and wash it with PBS (pH7.2) for 3 times, add FITC-labeled goat anti-mouse IgG Antibody, placed in a 37°C incubator for 2 hours, took out the cover slip and washed it with PBS (pH7.2) for 3 times, blotted the edge of the water and stained it with Evans blue, observed under a fluorescent microscope, MA after transfection of the recombinant expression vector -104 cells showed green fluorescence, and the results showed that the constructed recombinant expression vectors pVAXI- 43ku and pVAXI- 45ku could be expressed in mammalian cells, as shown in Figure 5.

Example 4 Combined immunization with pVAXⅠ- 43ku and pVAXⅠ- 45ku and its effect detection

(1) Immunization program

Fifteen BALB/C mice aged 4-5 weeks were randomly divided into 5 groups with 5 mice in each group. The groups and treatments of the experimental mice are shown in Table 1.

Table 1 Grouping and treatment of experimental mice

The inoculum concentration of groups B, C and D was 1 μg/μL, and group E was an equal-volume mixture of 1 μg/μL pVAXⅠ- 43ku and 1 μg/μL pVAXⅠ- 45ku .

The way of immunization was intramuscular injection of the hind limbs of the mice, once a week, for three consecutive weeks, and the next day after the last injection, the worms were challenged, with a dose of 2000 mice/mouse, and the way of oral gavage was adopted once.

(2) Detection of immune effect

Four weeks after attacking the worms, the mice were slaughtered, all the muscles were taken, and the rate of worm reduction was detected by conventional methods; the calculation formula was:

The reduction rate = the number of muscle larvae in the control group - the number of muscle larvae in the recombinant plasmid group/the number of muscle larvae in the control group × 100%.

The results showed that after immunization with pVAXⅠ- 43ku and pVAXⅠ- 45ku combined with DNA vaccine, the worm reduction rate could reach 76.65%, as shown in Table 2.

Table 2. The number of worms and the rate of worm reduction in each group

 

The tongue tissue was taken for HE staining and histopathological detection. The results showed that the number of worm eggs in the tongue tissue of group E mice was significantly less than that of the other 4 groups, as shown in Figure 6.

110> Jilin Agricultural University

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Claims (2)

1. Encoding ES Antigen from Trichinella spiralis Muscle Larvae gene vaccine, it comprises: recombinant plasmid pVAX I- 43kuwith pVAX I- 45ku, pVAX I- 43kuwith pVAX I- 45kuit is the gene inserted in carrier for expression of eukaryon pVAX I respectively as shown in sequence table SEQ ID No.1 and 2; Recombinant plasmid pVAX I- 43kuwith pVAX I- 45kuweight ratio be 1: 1; Concentration is 1 μ g/ μ L.

2. Encoding ES Antigen from Trichinella spiralis Muscle Larvae gene vaccine preparation method, comprising:

(1) prepare cultivation of larvae of Trichinella spiralis from muscle total serum IgE, reverse transcription is cDNA, and is template with cDNA, with primer:

43ku -P1：5’-CGCGGTACCATGCGAATATACATTTTTCTTAG-3’

43ku -P2：5’-CGAGGATCCTTAGCTGTATGGGCAA-3’

45ku -P1：5’-CGCGGTACCATGAAACTCTTGCTTTTAACA-3’

45ku -P2：5’-GCGGATCCTTAGCCTTGCTTAGAGAG-3’

PCR method increases 43ku, 45ku ES antigen gene respectively, and its base sequence of 43ku, 45ku gene is as shown in sequence table SEQ ID No.1,2;

(2) will 43ku, 45kugene inserts in pVAX I carrier for expression of eukaryon respectively, build recombinant eukaryon expression vector pVAX I- 43kuwith pVAX I- 43ku;

(3) two kinds of recombinant eukaryon expression vectors are proceeded to respectively e.coliin DH5 α, be inoculated in LB liquid nutrient medium, 37 DEG C, 150-180 r/min shaking table is cultivated;

(4) from above-mentioned cultivation e.colia large amount of preparation pVAX I in DH5 α- 43kuwith pVAX I- 45ku, and after both adjusting, concentration is 1 μ g/ μ L, by weight 1: 1 mixing.