CN102335422A - Recombinant phage peculiar smell removal vaccine for boars - Google Patents

Abstract

The present invention provides a boar odor-removing recombinant phage vaccine. The vaccine uses recombinant T7 bacteriophage as an antigen, and the surface of the recombinant T7 bacteriophage expresses a GnRH protein whose amino acid sequence is shown in SEQ ID NO: 2. Said GnRH protein is obtained by inserting three copies of GnRH gene into the downstream expression of T7 phage P10B gene. The present invention has the following technical effects: (1) the recombinant phage vaccine prepared with T7 phage carrier can overcome the weak self-immunogenicity of GnRH peptide hormones, effectively improve immunogenicity, stimulate the body to produce higher levels of antibodies, inhibit Development of the boar testes. (2) Since T7 bacteriophage is easy to cultivate and purify, it has multiple advantages in the production of genetically engineered vaccines, and the recombinant phage deodorizing vaccine prepared by the present invention is convenient for large-scale production and low in cost. (3) The immunological method is more humane, thereby replacing the traditional castration method and meeting the needs of modern large-scale breeding and management.

Description

Boar odor-removing recombinant phage vaccine

technical field

The invention relates to a boar odor-removing recombinant phage vaccine which removes boar endogenous odor substances and improves meat quality by immunological methods, and belongs to the field of molecular biology technology.

Background technique

GnRH is synthesized by GnRH neurons in the hypothalamus and stored in the cells in the form of granules or vesicles. Thirteen different GnRHs have been isolated from the nerve tissues of vertebrates and primordial animals. The GnRH structures of all mammals including humans It is the same, its amino acid sequence is: PYROGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, that is, pyroglutamic acid-histidine-tryptophan-serine-tyrosine -Glycine-Leucine-Arginine-Proline-Glycyl Peptide. After active immunization of animals with GnRH vaccine, the animal body can be induced to produce specific anti-GnRH antibodies, which bind to the endogenous GnRH secreted by the animal itself and make it lose its biological activity, thus leading to the function of the hypothalamus-pituitary-gonadal axis The destruction of LH and FSH inhibits the release of LH and FSH, which in turn leads to changes in gonadal endocrine. For male animals, it leads to decreased secretion of steroid hormones, atrophy of testes and gonads, and obstruction of sperm production; for female animals, it leads to prolonged anestrus or no estrus, obstruction of follicle development, ovulation, and corpus luteum formation, and atrophy of ovaries and uterus. Ultimately, most or even complete loss of reproductive capacity of animals.

In most livestock, non-castrated males grow faster, more efficiently, and have better ketone body quality than castrated males. Wood et al. found that uncastrated boars grew faster and paid more for feed than castrated boars. Kuhlers et al. reported that uncastrated boars had significantly larger eye muscles compared to castrated boars of the same body weight. Robertson et al. used GnRH to immunize bulls and found that the testicular atrophy of the immunized bulls, the "castration" effect of reducing libido lasted for 6 months, and its fat decreased by 32%, and the eye muscle area at the 10th rib increased by 50%. 12% lower than the ratio.

In theory, producing uncastrated boars can bring obvious economic benefits, but there are still many unfavorable factors to be overcome in the actual production process. Uncastrated boars are obviously aggressive after sexual maturity, and have higher requirements for breeding facilities, and need to be reared separately by gender, and the danger to breeders is also significantly increased. Sexually mature boars climb frequently, and pigs bite and fight more, which not only increases the probability of injury but also reduces feed remuneration, which brings great inconvenience to the production process. Another problem with producing castrated boars is the unpleasant taste that this pork develops during cooking, colloquially known as 'pig odour'. Studies have shown that 5%-35% of ketone bodies in uncastrated boar pork have an unpleasant smell for consumers. The peculiar smell of boars appears for the first time during the estrus period, and gradually increases when the boar reaches 70kg body weight or 4 months of age. Most of the odorous substances in fat are uncastrated pigs with a body weight greater than 95kg or a growth period greater than 5 months Boars, but will gradually disappear after castration, and their sexual behavior will gradually disappear after castration and become docile. In the process of pig breeding in my country, surgical castration is often used to deal with the peculiar smell in pork. This method is simple and thorough, but it also brings many negative effects. The testicles of pigs are highly tubular and full of nerves, and surgical castration can only be performed on immature young pigs, not on sexually mature older pigs. At the same time, surgical castration not only reduces the productivity and meat quality of pigs, but also has many risks: wound bleeding, infection, severe hernia, stunting or death of animals.

In recent years, various methods have been used to try to overcome the disadvantages of surgical castration, for example, implanting hormones in the body and chemical castration. Hormone treatment requires repeated injections, and chemical castration can sterilize but affect the growth performance of pigs. Therefore, there is a need for a method that can suppress the off-flavor substances in pork and does not affect the growth performance of pigs. The immunocastration method involved in the present invention can well take into account the needs of the two.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of the prior art and provide a boar odor-removing recombinant phage vaccine.

The boar odor-removing recombinant phage vaccine of the present invention uses recombinant T7 phage as an antigen, and the surface of said recombinant T7 bacteriophage expresses an amino acid sequence such as GnRH protein shown in SEQ ID NO: 2, and said GnRH protein consists of three parts A copy of the GnRH gene was inserted into the downstream expression of the T7 phage P10B gene.

The GnRH gene of the three copies is selected from the nucleotides of the following sequences:

1) have the nucleotide sequence shown in SEQ ID NO: 1;

2) a nucleotide sequence having an increased or decreased copy number of the sequence shown in SEQ ID NO: 1;

3) Have the sequence shown in SEQ ID NO: 1 change, delete or increase one or several nucleotides but still express the nucleotide sequence with GnRH main antigen activity polypeptide.

Said vaccine also contains white oil adjuvant for emulsifying recombinant T7 phage.

The construction method of boar odor-removing recombinant phage vaccine of the present invention comprises the following steps:

A. Construction of recombinant phage

(1) Synthesize a DNA sequence, which is cloned into the multiple cloning site of the pUC-19 vector. The 5' end of the DNA sequence has a restriction site EcoRI, and the 3' end contains a restriction site HindⅢ. The DNA sequence is as follows: Shown in SEQ ID NO: 3;

(2) Excise the above DNA sequence from the pUC-19 vector with restriction endonucleases EcoRI and HindIII and insert it between EcoRI and HindIII of the T7 phage multiple cloning site to construct a recombinant phage;

(3) T7 phage packaging protein was used to assemble the recombinant phage, the packaging product was amplified by the agarose sandwich method, and the positive recombinant phage was screened by PCR method, and the recombinant phage displayed 3 copies of the GnRH protein, and its amino acid sequence was shown in SEQ ID NO: 2 ;

B. Preparation of recombinant phage

E. coli BL21 strain cryopreserved in glycerol was inoculated by streaking on the plane of LB medium, and cultivated overnight at 37°C;

Pick a single colony from the plate, inoculate 5mL of LB culture solution, culture overnight at 37°C and 200 rpm with shaking, take 3mL of the overnight culture and inoculate 300mL of LB culture solution, cultivate to about OD ₆₀₀ =0.8, pick a single colony on the plate Phage plaques, inoculated into the cultured BL21 host bacteria, 37 ° C, 100 rpm shaking culture for 2-3 hours, until the bacterial liquid changed from turbid to clear, recovered the phage by PEG precipitation, and followed the conventional method Measure the phage titer, adjust the recovered phage concentration to 2×10 ¹³ pfu/mL, add formaldehyde solution at a ratio of 4‰, shake and inactivate overnight at 37°C and 100 rpm;

C. Preparation of recombinant phage oil emulsion vaccine

1) Preparation of vaccine oil phase: 4mL Siben 80, 2mL Siben 85, 94mL No. 10 mineral oil, 2g aluminum stearate, mix thoroughly, and pressurize at 121°C for 20 minutes; preparation of vaccine water phase: 94mL titer is 2×10 ¹³ pfu/mL of inactivated bacteriophage, 4 mL of autoclaved Tween-80, 3000 rpm and stir well;

2) Emulsify to a stable water-in-oil structure on a high-speed emulsifier according to the water phase:oil ratio of 1:3, which is the prepared recombinant phage oil emulsion vaccine;

In the construction method of the boar odor-removing recombinant phage vaccine, the carrier of the recombinant T7 phage is T7 Select 415-1b.

The present invention has following technical effect:

(1) The present invention utilizes the characteristics of the adjuvant of the virus-like particle that the phage has, and the recombinant phage vaccine prepared with the T7 phage carrier can overcome the weak self-immunogenicity of the GnRH peptide hormone, effectively improve the immunogenicity, and stimulate the body Produce higher levels of antibodies, inhibit the development of testes in boars, and control the production of off-flavor substances in pork.

(2) Since T7 bacteriophage is easy to cultivate and purify, it has multiple advantages in the production of genetically engineered vaccines, and the recombinant phage deodorizing vaccine prepared by the present invention is convenient for large-scale production and low in cost.

(3) The immunological method is more humane, thereby replacing the traditional castration method and meeting the needs of modern large-scale breeding and management.

Description of drawings

Figure 1 is the gene identification of recombinant T7 phage. Lane 1 is DL2000 DNA Marker; Lane 2 is T7 phage vector control; Lane 3 is recombinant T7 phage.

Fig. 2 is a diagram showing the level of GnRH antibody in boars immunized with recombinant phage deodorizing vaccine.

Fig. 3 is a diagram showing testosterone levels of boars after immunization with recombinant phage deodorizing vaccine.

Fig. 4 is a diagram showing the size of boar double testis after immunization with the recombinant phage deodorizing vaccine.

Figure 5-1 is a diagram of boar testis section after immunization with recombinant phage deodorizing vaccine.

Figure 5-2 is a sliced view of testes of uncastrated boars.

Detailed ways

In the embodiment,

The gene encoded as shown in SEQ ID NO: 3 is the main epitope of GnRH and is a conventional technology.

Restriction endonucleases EcoRI and HindⅢ were purchased from Dalian Bao Biological Engineering Company.

T7 phage vector, T7 packaging protein, and E.coli BL21 were purchased from Novagen.

The extraction of T7 phage is a routine technique.

The determination of T7 phage titer is a routine technique.

Example 1 Construction of recombinant phage

A DNA sequence was synthesized by a commercial company. The DNA sequence has been cloned into the multiple cloning site of the pUC-19 vector. The 5' end of the DNA sequence has a restriction site EcoRI, and the 3' end contains a restriction site HindⅢ. The DNA sequence As shown in SEQ ID NO: 3

The above DNA sequence was excised from the pUC-19 vector with restriction endonucleases EcoRI and HindIII and inserted between EcoRI and HindIII of the T7 phage multiple cloning site to construct a recombinant phage.

Recombinant pUC-19 vector double enzyme digestion system:

ddH ₂ O 16.0 μL 10×H Buffer 5.0 μL pUC-19 vector 25.0 μL EcoRI 2.0 μL HindⅢ 2.0 μL

Mix the above reaction system and place it in a 37°C water bath for 4 h. After the digested products are also identified by 1% agarose gel electrophoresis, the gel recovery kit is used for gel recovery and identification.

T7 phage vector double enzyme digestion system:

ddH2O 11.0 μL 10×T Buffer 2.0 μL T7-Select 415b carrier 5.0 μL EcoRI 1.0 μL HindⅢ 1.0 μL

Mix the above reaction system and place it in a 37°C water bath for 4 hours. After the digested products are also identified by 0.5% agarose gel electrophoresis, the gel recovery kit is used for gel recovery and identification.

Connection reaction system:

DNA fragment recovery product 2.0 μL T7-Select 415b carrier recovery product 0.5 μL ddH2O 1.75 μL T4 DNA Ligase 0.25 μL 10×T4 DNA Ligase Buffer 0.5 μL

Ligation overnight at 16°C.

Packaging of recombinant phage:

Ligation product 5.0 μL T7 Packing Extracts 25.0 μL

Pack at 22°C for 2 hours, add 270 μL LB medium to the packaging reaction system to terminate the reaction.

Plate amplification of packaging products:

Mix 100 μL of packaging reaction with 250 μL of fresh overnight cultured BL21 bacterial solution (OD600=1.0), then quickly mix with 3 mL of melted top layer agar in a 45°C warm bath and spread evenly on LB plates. After the top layer of agar solidifies, culture it upside down at 37°C to form plaques. The plaques were picked, and the positive recombinant phages were screened by PCR method. The GnRH protein is displayed on the surface of the recombinant phage, and its amino acid sequence is shown in SEQ ID NO:2.

Example 2 Large-scale amplification of recombinant phage

E. coli BL21 strain cryopreserved in glycerol was inoculated by streaking on the plane of LB body medium, and cultivated overnight at 37°C; a single colony was picked from the plate, inoculated with 5 mL of LB medium, and cultured overnight at 37°C and 200 rpm with shaking. Take 3 mL of the overnight culture and inoculate 300 mL of LB culture medium, and cultivate to about OD ₆₀₀ =0.8. Pick a single phage plaque on a plate, inoculate it into the cultured BL21 host bacteria, and incubate with shaking at 37°C and 100 rpm for 2-3 hours until the bacterial solution changes from turbid to clear. The phage was recovered by PEG-NaCl precipitation, and the phage titer was determined according to the conventional method. The recovered phages were adjusted to a concentration of 2×10 ¹³ pfu/mL, added to formaldehyde solution at a ratio of 4‰, and shaken at 37°C and 100 rpm overnight for inactivation.

Example 3 Preparation of phage deodorant oil emulsion vaccine

The preparation method of recombinant phage deodorizing oil emulsion vaccine is as follows:

Preparation of the oil phase of the vaccine: 4mL Siben 80, 2mL Siben 85, 94mL No. 10 mineral oil, 2g aluminum stearate, mix well, and press at 121°C for 20 minutes under high pressure. Preparation of vaccine aqueous phase: 94 mL of inactivated phage with a titer of 2×10 ¹³ pfu/mL, 4 mL of autoclaved Tween-80, stirred at 3000 rpm. According to the ratio of water phase: oil ratio of 1:3, add 150mL oil phase to the tissue masher, slowly add 50mL water phase while stirring slowly, after mixing well, stir at 10000 rpm/quickly for 2 minutes until a stable The water-in-oil structure is the prepared recombinant phage oil emulsion vaccine, which is stored at 4°C for later use.

Example 4 Recombinant phage deodorizing oil emulsion vaccine immunized boars and its effect

The above-mentioned recombinant phage deodorizing oil emulsion vaccine is immunized for the first time when the boar is 9 weeks old, and then immunized for the second time after an interval of 4-9 weeks, and the boar can be slaughtered 4-5 weeks after the second immunization. The antigen content in the recombinant T7 phage vaccine reached 5×10 ¹² pfu/mL, and each pig was immunized with 2 mL each time. The specific operation is as follows:

Three-way hybrid boars were selected as experimental animals. Select 50 healthy boars of similar age at birth, 10 of which were surgically castrated 3 days after birth, and the remaining pigs were kept for observation until 9 weeks old, and 2 mL of the vaccine was injected subcutaneously in the neck to immunize 30 boars in total. 10 heads were used as blank control. After immunization, observe the sexual behavior and the development of double testes in the pig herd. Surgical castration can be performed on a very small number of pigs whose first immunization fails (normal double testes development) due to individual differences. According to the growth rate of the pig herd and the slaughter time, within 4 to 9 weeks after the first immunization, the same dose is used for the second immunization on the same part. Pigs can be slaughtered 4 to 5 weeks after the second immunization.

After immunization, blood samples were collected every week to detect the GnRH antibody and testosterone levels in the serum, and testicular development was measured with a vernier caliper. Some pigs were slaughtered 5 weeks after the second immunization, and the testes were collected for weighing, size measurement, sperm presence and sperm motility, and back fat was collected to detect androstenone content. The testicular tissue was fixed in formalin solution, and tissue sections were made to analyze the morphology of testicular tissue.

As shown in Figure 2, after the recombinant phage immunization, the body produced a high level of anti-GnRH antibody, and the antibody reached the peak two weeks after the second immunization, and the binding rate with GnRH reached more than 80%.

As shown in Figure 3, the testosterone level of the immunized group decreased rapidly after the first immunization, and the testosterone level of the non-castrated boars gradually increased with age until it stabilized at a high level of 8 pmol/mL in the late growth period; The testosterone level dropped below 4 pmol/mL when the concentration reached, and the testosterone level after booster immunization was equivalent to that of the surgical castration control.

As shown in Figure 4, the development of the double testes in the immunized group was inhibited and basically maintained at 4 cm in size; the testes of uncastrated boars gradually increased with growth and development, and reached 10 cm before slaughter.

As shown in Figure 5-1, the spermatogenic cells in the immunized group were sparsely arranged and there were fewer sperm; as shown in Figure 5-2, the spermatogenic cells in the non-castrated control group were closely arranged and there were more sperm in the cavity.

<110> OrganizationName : Jiangsu Academy of Agricultural Sciences

the

Application Project

-------------------

<120> Title : Recombinant bacteriophage vaccine for deodorizing boars

<130> AppFileReference :

<140> CurrentAppNumber :

<141> CurrentFilingDate : ____-__-__

the

sequence

--------

<213> OrganismName :

<400> PreSequenceString :

gaattcgcaa cattggtctt atggtttacg tcctggtcag cactggtcct acggcttgcg 60

cccaggccaa cattggtcat atggtttacg tcctggttaa aagctt 106

<212> Type : DNA

<211> Length : 106

SequenceName : 1

SequenceDescription :

the

sequence

--------

<213> OrganismName :

<400> PreSequenceString :

EHWSYGLRPG EHWSYGLRPG EHWSYGLRPG 30

<212> Type : PRT

<211> Length : 30

SequenceName : 2

SequenceDescription :

the

sequence

--------

<213> OrganismName :

<400> PreSequenceString :

gaattcggac gacgacaagc aacattggtc ttatggttta cgtcctggtc agcactggtc 60

ctacggcttg cgcccaggcc aacattggtc atatggttta cgtcctggtt aaaagctt 118

the

<212> Type : DNA

<211> Length : 118

SequenceName : 3

SequenceDescription :

the

Claims (5)

1. boar removes abnormal flavour recombinant phage vaccine; It is characterized in that; Biting the T7 thalline with reorganization is antigen; Said reorganization T7 phage surface is expressed has the GnRH albumen of aminoacid sequence shown in SEQ ID NO:2, and said GnRH albumen inserts the downstream expression acquisition of T7 phage P10B gene by the GnRH gene of three parts of copies.

2. boar according to claim 1 removes abnormal flavour recombinant phage vaccine, it is characterized in that, the GnRH gene of described three parts of copies is selected from the nucleotide of following sequence:

1) has nucleotide sequence shown in the SEQ ID NO:1;

2) has sequence shown in the SEQ ID NO:1 through increasing or reduce the nucleotide sequence of copy number;

3) has sequence shown in the SEQ ID NO:1 through changing, lack or increasing one or several nucleotide but still express nucleotide sequence with GnRH major antigen active polypeptide.

3. boar according to claim 1 removes abnormal flavour recombinant phage vaccine, it is characterized in that, also comprises the white-oil adjuvant that is used for emulsifying reorganization T7 phage in the said vaccine.

4. any one boar removes the construction method of abnormal flavour recombinant phage vaccine among the claim 1-3, it is characterized in that, comprises the following steps:

A, structure recombinant phage

(1) synthetic section of DNA sequence, this DNA sequence is cloned in the pUC-19 carrier MCS, and 5 ' end of DNA sequence has restriction enzyme site EcoR I, and 3 ' end contains restriction enzyme site Hind III, and its DNA sequence is shown in SEQ ID NO:3;

(2) above-mentioned DNA sequence is downcut the back from the pUC-19 carrier insert between the EcoR I and Hind III of T7 phage MCS with restricted enzyme EcoR I, Hind III, make up recombinant phage;

(3) with T7 phage packaging albumen assembling recombinant phage, the packing product screens positive recombinant phage through the amplification of agarose sandwich assay with PCR method, and recombinant phage is showed the GnRH albumen of 3 copies, and its aminoacid sequence is shown in SEQ ID NO:2;

The preparation of B, recombinant phage

The E. that glycerol is frozen ColiThe streak inoculation on LB body culture medium plane of BL21 bacterial strain, 37 ℃ of incubated overnight; Picking list bacterium colony from the plate, inoculation 5mL LB culture fluid, 37 ℃, 200 rev/mins concussion overnight incubation are got 3mL overnight culture inoculation 300mL LB culture fluid, are cultured to OD ₆₀₀About=0.8; Single plaque on the picking plate is inoculated in the cultured BL21 host bacterium, and 37 ℃, 100 rev/mins concussions were cultivated 2-3 hour; Become clarification until bacterium liquid by muddiness; Reclaiming phage with the sedimentary method of PEG, and measure phage titre by the method for routine, is 2 * 10 with the phage adjustment concentration that reclaims ¹³Pfu/mL, and add formalin according to 4 ‰ ratio, 37 ℃, 100 rev/mins concussion deactivations are spent the night;

C, preparation recombinant phage oil emulsion vaccine.

5. boar according to claim 1 removes abnormal flavour recombinant phage vaccine, it is characterized in that, the carrier of described reorganization T7 phage is T7 Select 415-1b.

Images