CN117721131A - Preparation method and application of recombinant bone morphogenetic protein mutant - Google Patents

Abstract

The invention is applicable to the field of biotechnology and provides a preparation method and application of a recombinant bone morphogenetic protein mutant. The preparation method of the recombinant bone morphogenetic protein mutant includes: constructing an inactive recombinant bone morphogenetic protein mutant, recombinant Prokaryotic expression and purification of the protein; application steps include: immunizing mice with the recombinant protein and evaluating its immune protective effect against Trichinella spiralis infection. The recombinant bone morphogenetic protein mutant in the present invention can induce mice to produce high-titer serum-specific antibodies, and exhibits good immunoprotective effects against subsequent Trichinella infection. The present invention applies the recombinant bone morphogenetic protein mutant in the study of immune protective effects against Trichinella infection and the preparation of related vaccines, which is of great significance in preventing Trichinella infection.

Description

Preparation method and application of a recombinant bone morphogenetic protein mutant

Technical field

The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of a recombinant bone morphogenetic protein mutant.

Background technique

Trichinella spiralis is a multicellular parasite that lives a parasitic life. It can infect a variety of animals and live in the muscle cells of animals for a long time. When humans or animals eat meat products containing infective muscle larvae, Trichinella spiralis is released through the digestion of pepsin. Trichinella spiralis can invade the small intestinal epithelium within 6 hours of infection, where it undergoes four molts to develop into adult worms. Adult worms begin to produce new larvae 5 days after infection, which continues until the adult worms are eliminated from the body. Newborn larvae can invade the host's muscle cells with the help of blood circulation or lymph circulation and establish long-term infection. The three developmental stages of Trichinella spiralis are all in the same host and overlap in time, which is its unique parasitic mode. The survival time of intestinal adult worms and the successful migration rate of newborn larvae are the keys to successful parasitism of muscle larvae. Therefore, the main stages of research on reducing the risk of Trichinella infection from the perspective of preventing and controlling Trichinella infection and preparing vaccines should focus on the intestinal adult stage and the migration stage of newborn larvae. At present, the vaccine research points for Trichinella spiralis include: natural antigens of the parasite, recombinant antigens and DNA vaccines. Considering that natural antigens are difficult to prepare and have strong unsafe factors, the application of their vaccines is somewhat difficult. The recombinant single protein has simple ingredients, perfect preparation technology, and low biosafety risks, making it one of the important directions for trichinella vaccine research. Screening key antigenic components from the parasitic process of Trichinella spiralis is the direction of recombinant protein vaccine research.

Bone morphogenetic protein (BMP) is a member of the transforming growth factor protein (TGF-β) superfamily, which can transmit signals to the interior of cells through serine-threonine protein kinase receptors. BMP has the typical synthesis, secretion and activation characteristics of TGF-β protein. It is synthesized in cells and cleaved by enzymes to form two parts that rely on non-covalent bonding. The two BMPs form a stable, protein-free The dimer complex is subsequently released outside the cell. Outside the cell, it can be stimulated by integrins and release active dimers, which act on receptors on the surface of target cells, causing biological effects. BMP participates in various stages of animal growth and development, including: iron metabolism, osteoblast differentiation, embryonic differentiation, etc. The transformation factor protein of Trichinella spiralis ( Ts -Tgh4) belongs to the BMP family and plays an important role in the growth and development of Trichinella spiralis. Therefore, we predicted the activation site of Ts -Tgh4 and obtained protein mutants, and evaluated Ts -Potential value of Tgh4 mutants as vaccine candidates. To this end, we propose a recombinant bone morphogenetic protein mutant and its preparation method and application.

Contents of the invention

The purpose of the present invention is to provide a preparation method and application of a recombinant bone morphogenetic protein mutant, aiming to solve the problems raised in the above background technology.

In order to achieve the above objects, the present invention provides the following technical solutions:

A method for preparing a recombinant bone morphogenetic protein mutant, including the following steps:

Step 1: Combine the website protein structure prediction and amino acid multiple sequence comparison results to predict the key amino acid sites for the biological activity of bone morphogenetic protein, mutate the corresponding bases, and obtain the mutated gene sequence;

Step 2. Construct an expression plasmid: Select the XhoI and NdeI endonuclease sites, clone the original gene and the mutated gene sequence into the Pet28a vector, then transform it into the E. coli DH5a competent strain, and pass it through containing Canaryola sulfate The positive single-clonal strains were screened out on the antibiotic-resistant LB culture plate, and then the plasmids of the single-clonal strains were sequenced and identified; the identified positive clones were expanded and cultured, the plasmids were extracted, and the plasmids were transformed into E. coli BL21 (DE3) and screened again. Positive single clone strains were identified and the strains were sequenced and identified;

Step 3. Express recombinant protein: Cultivate the positive monoclonal strain obtained in Step 2 in a shaker at 37°C and 120 rpm. When the absorbance of the culture solution reaches 0.4-0.6, add a final concentration of 0.1 nmol. Isopropyl-β-D-thiogalactopyranoside (IPTG) inducer, continue to culture for 6-8 hours, centrifuge to collect E. coli and use PBS buffer to wash E. coli; ultrasonically disrupt the collected E. coli and collect Inclusion bodies that are insoluble in water are dissolved with 8 mol of urea;

Step 4. Purify the recombinant protein: Use ÄKTA pure fully automatic protein separation and purification instrument to purify and separate the inclusion bodies. The recombinant protein with His tag binds non-specifically to the nickel column and is competitively eluted by imidazole, thereby obtaining the purified recombinant protein. protein.

A recombinant bone morphogenetic protein mutant prepared by the above preparation method.

Application of the above recombinant bone morphogenetic protein mutant in the preparation of preventive preparations against Trichinella spiralis infections.

Further steps include:

Step 5. Immunize mice with recombinant protein;

Step 6: Evaluate the immune protective effect of the recombinant protein against Trichinella spiralis infection.

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

1. The present invention proposes a protein mutation scheme for bone morphogenetic protein. By mutating key amino acids, the functional activity of the protein is lost, and a single recombinant protein is obtained by prokaryotic expression and purification. Mice were immunized using a mixture of recombinant bone morphogenetic protein and Freund's adjuvant. After three immunization procedures 14 days apart, the mice were induced to produce high-level antibody titers. Mice were orally infected with Trichinella spiralis. After a 35-day parasitic cycle, the reduction rate of the number of Trichinella spiralis in the muscles of the mice was identified as the immune protection of the recombinant protein. The results show that the recombinant bone morphogenetic protein mutant can induce mice to produce high titer antibody levels and good immune protection.

2. The present invention applies recombinant bone morphogenetic protein mutants in the preparation of anti-Trichinella infection prevention preparations; it is the first time that Trichinella spiralis-derived recombinant bone morphogenetic protein mutants and effective immunoprotective effects have been discovered, which can effectively prevent and treat trichinellosis. of great significance.

Description of the drawings

In Figure 1: A is the predicted site and mutation scheme of the key amino acids for Tgh-4 protein activity; B is the PCR amplification chart of the Tgh-4 protein mutant gene; C is the double endonuclease digestion of the two gene construction vectors. Identification diagram; D is the SDS-page diagram of the two purified proteins; E is the sequencing diagram of the constructed vector.

In Figure 2: A is a schematic diagram of the protein immunization procedure and immune protection detection time points; B is the antibody titer of protein-specific IgG in the 35-day serum; C is the hematoxylin-eosin staining picture of the diaphragm; D is the small Statistical chart of the total number of worms and the number of dead worms in mice.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention more clear, the present 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 here are only used to explain the present invention and are not intended to limit the present invention.

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

One embodiment of the present invention provides a method for preparing a recombinant bone morphogenetic protein mutant, including the following steps:

Step 1: Combine the website protein structure prediction and amino acid multiple sequence comparison results to predict the key amino acid sites for the biological activity of bone morphogenetic protein, mutate the corresponding bases, and obtain the mutated gene sequence; the mutation scheme is shown in Figure 1 Shown in A.

Step 2. Construct an expression plasmid: Select the XhoI and NdeI endonuclease sites, clone the original gene and the mutated gene sequence into the Pet28a vector, then transform it into the E. coli DH5a competent strain, and pass it through containing Canaryola sulfate The positive single-clonal strains were screened out on the antibiotic-resistant LB culture plate, and then the plasmids of the single-clonal strains were sequenced and identified; the identified positive clones were expanded and cultured, the plasmids were extracted, and the plasmids were transformed into E. coli BL21 (DE3) and screened again. Positive single clone strains were obtained, and the strains were sequenced and identified; the constructed plasmid was verified by endonuclease digestion, and the results are shown in Figure 1 B-C.

Step 3. Express recombinant protein: Cultivate the positive monoclonal strain obtained in Step 2 in a shaker at 37°C and 120 rpm. When the absorbance of the culture solution reaches 0.4-0.6, add a final concentration of 0.1 nmol. IPTG inducer, continue culturing for 6-8 hours, centrifuge to collect the E. coli and use PBS buffer to wash the E. coli; ultrasonically disrupt the collected E. coli, collect water-insoluble inclusion bodies, and dissolve the inclusion bodies with 8 mol of urea;

Step 4. Purify the recombinant protein: Use the ÄKTA pure fully automatic protein separation and purification instrument to purify and separate the inclusion bodies. The recombinant protein has a His tag, which can non-specifically bind to the nickel column and can be competitively eluted by imidazole, thereby obtaining the purified protein. of recombinant proteins. SDS-page analysis of recombinant protein is shown in D in Figure 1.

One embodiment of the present invention provides a recombinant bone morphogenetic protein mutant prepared by the above preparation method.

One embodiment of the present invention provides an application of the above-mentioned recombinant bone morphogenetic protein mutant in the preparation of anti-Trichinella infection prevention preparations.

As a preferred embodiment of the present invention, it includes the following steps:

Step 5. Immunize mice with recombinant protein;

Step 6: Evaluate the immune protective effect of the recombinant protein against Trichinella spiralis infection.

As a preferred embodiment of the present invention, step five specifically includes:

Mix the recombinant protein with an equal volume of Freund's adjuvant, stir thoroughly until the mixture is insoluble in water, inoculate each mouse with a dose of 50 μg/100 μl of the mixture, and immunize by intraperitoneal injection; use complete Freund's adjuvant for the first immunization It can more effectively induce an active immune response. Incomplete Freund's adjuvant is used for the second and third booster immunizations; the first immunization is on day 0, the second and third immunizations are on days 14 and 28 respectively; the third immunization is One week after the end, that is, on the 35th day, the serum of the mice was collected and the specific antibody level of the recombinant protein in the serum was detected. Then, 250 Trichinella spiralis were infected by gavage. On the 70th day, the number of intramuscular larvae of the mice was counted. quantity. The embodiment is shown as A in Figure 2.

As a preferred embodiment of the present invention, step six specifically includes:

Detect the specific antibody level of the recombinant protein in the serum: use 100 μl coating solution to dilute 0.5 μg of the recombinant protein and embed it in a 96-well plate, and incubate at 4°C overnight; wash three times with PBST, and then block with 5% BSA blocking solution at 37°C. 1 hour; wash three times with PBST, add diluted serum and incubate at 37°C for 1 hour; wash three times with PBST, add dilute horseradish peroxidase-conjugated goat anti-mouse IgG antibody; wash three times with PBST, add 100 μl of TMB chromogenic substrate was used to develop color for 10 minutes, and then 50 μl of stop solution was added to terminate the reaction. A multifunctional microplate reader was used to detect the absorbance at 450 nm, and the specific antibody level of the recombinant protein in the serum was calculated; the results are shown in B in Figure 2 Show.

To evaluate the immunoprotective effect: the mice were sacrificed and their diaphragms and muscle tissues were collected. Muscle tissue was fixed in formalin and stained with hematoxylin-eosin. Prepare digestive juice containing 1% hydrochloric acid and 1% pepsin to digest the remaining minced muscle in a 37°C incubator for 2 hours. Then the digested liquid is washed and settled, and the upper liquid is discarded. After multiple washings, the number of Trichinella spiralis that sinks to the bottom is counted and the infection rate is calculated. The results are shown in D in Figure 2.

The above are only the preferred embodiments of the present invention. It should be pointed out that those skilled in the art can also make several modifications and improvements without departing from the concept of the present invention, and these should also be regarded as the protection scope of the present invention. , none of these will affect the effect of the present invention and the practicality of the patent.

Claims (4)

1. A method for preparing a recombinant bone morphogenic protein mutant, comprising the steps of:

step one, combining the website protein structure prediction and amino acid multi-sequence comparison results, predicting key amino acid sites of the biological activity of the bone morphogenetic protein, and mutating corresponding bases to obtain a mutated gene sequence;

step two, constructing an expression plasmid: selecting XhoI and NdeI endonuclease sites, cloning an original gene and a mutated gene sequence into a Pet28a carrier, then converting the original gene and the mutated gene sequence into escherichia coli DH5a competence, screening out a positive monoclonal strain by an LB culture plate containing kanamycin sulfate resistance, and then carrying out sequencing identification on plasmids of the monoclonal strain; amplifying and culturing the identified positive clone, extracting plasmid, transforming the plasmid into escherichia coli BL21 (DE 3), screening out positive monoclonal strain again, and carrying out sequencing identification on the strain;

step three, expressing recombinant protein: culturing the positive monoclonal strain obtained in the second step, culturing in a shaking table at 37 ℃ and 120 r/min, adding IPTG inducer with the final concentration of 0.1nmol when the absorbance of the culture solution reaches 0.4-0.6, continuously culturing for 6-8 hours, centrifuging to collect the escherichia coli, and washing the escherichia coli by using PBS buffer solution; carrying out ultrasonic crushing on the collected escherichia coli, collecting inclusion bodies insoluble in water, and dissolving the inclusion bodies with 8mol of urea;

step four, purifying recombinant protein: purifying and separating the inclusion body by using a Ä KTA pure full-automatic protein separation and purification instrument, wherein the recombinant protein with the His tag is non-specifically bound to a nickel column and is subjected to competitive elution by imidazole, so that the purified recombinant protein is obtained.

2. A recombinant bone morphogenic protein mutant produced according to the method of claim 1.

3. Use of a recombinant bone morphogenic protein mutant according to claim 2 for the preparation of a prophylactic preparation against trichina infection.

4. The use according to claim 3, characterized by the following steps:

step five, immunizing mice by recombinant proteins;

and step six, evaluating the immune protection effect of the recombinant protein on the infection of the trichina.