Mycoplasma gallisepticum RRF recombinant protein, polyclonal antibody and preparation method thereof
By preparing recombinant Mycoplasma gallisepticum RRF protein and expressing it in a prokaryotic system, and then combining it with animal immunization to prepare polyclonal antibodies, the problem of the lack of commercial antibodies was solved, and the specific detection and study of RRF protein was realized.
Patent Information
- Application Number
- CN202511454616.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-13
- Publication Date
- 2026-01-23
AI Technical Summary
The lack of commercially available polyclonal antibodies against Mycoplasma gallisepticum RRF in existing technologies makes it difficult to screen for novel drugs targeting RRF, and it is also difficult to detect the protein level of RRF in bacteria.
Recombinant Mycoplasma gallisepticum RRF protein was prepared, expressed and purified in a prokaryotic system using the pET-32a+ expression vector, and polyclonal antibodies were prepared by animal immunization. The specificity and reliability of the antibodies were verified by Western blotting.
A polyclonal antibody that specifically recognizes the RRF protein of Mycoplasma gallisepticum is provided for ELISA and Western Blot experiments, providing a material basis for RRF function research and ensuring the specificity and applicability of the antibody.
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Figure CN121378428A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, and more specifically to a recombinant Mycoplasma gallisepticum RRF protein, a polyclonal antibody, and a method for preparing the same. Background Technology
[0002] Ribosome recycling factor (RRF) is an important protein involved in the termination of protein synthesis. When the stop codon on mRNA reaches the aminoacyl (A) site of the ribosome and is recognized by the release factor, it promotes the release of the polypeptide chain, resulting in translation termination. The ribosome leaves mRNA and deacylated tRNA at the P and E sites. In bacteria, the 70S ribosome complex after translation termination needs to be recycled through the synergistic action of RRF and elongation factor G (EF-G). Ribosome recycling is a necessary step for the initiation of a new round of translation. This process requires the 70S ribosome to be split into 30S and 50S subunits, and this splitting process requires guanosine triphosphate (GTP) for energy.
[0003] Currently, the main research direction regarding RRF is exploring the molecular mechanisms of ribosome cycling. Low-level expression or absence of RRF in bacterial cells leads to irreversible bacterial death. Therefore, drugs targeting RRF would be a novel antibacterial mechanism.
[0004] However, mycoplasma genomes are among the smallest in prokaryotes, limiting their biosynthetic and metabolic capabilities. Using mycoplasma may allow for more targeted screening of novel drugs targeting RRF. This is especially true given the lack of commercially available polyclonal antibodies against RRF from the Mycoplasma Gallisepticum (MG) R strain. Further screening for novel drugs and investigating their mechanisms of action requires detecting RRF protein levels within the bacteria. The development of RRF antibodies could also aid in screening other lead compounds targeting RRF.
[0005] Therefore, how to develop a polyclonal antibody against Mycoplasma gallisepticum R strain RRF is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0006] In view of this, the purpose of the present invention is to provide a recombinant protein of Mycoplasma gallisepticum RRF, a polyclonal antibody and a method for preparing the same, so as to overcome the shortcomings of the prior art.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] A recombinant protein of Mycoplasma gallisepticum RRF, the amino acid sequence of which is shown in SEQ ID NO:1.
[0009] Furthermore, the three-letter amino acid sequence of the above-mentioned Mycoplasma gallisepticum RRF recombinant protein is shown in SEQ ID NO:2.
[0010] Furthermore, the full-length sequence of the above-mentioned Mycoplasma gallisepticum RRF recombinant protein is shown in SEQ ID NO:3.
[0011] Furthermore, the plasmid used to construct the recombinant protein vector is the pET-32a+ expression vector.
[0012] A method for preparing the above-mentioned Mycoplasma gallisepticum RRF recombinant protein specifically includes the following steps:
[0013] (1) Transform the recombinant protein vector into competent cells to construct engineered bacteria with the recombinant vector;
[0014] (2) The engineered bacteria were induced by IPTG to express the recombinant protein of Mycoplasma gallisepticum RRF and then purified to obtain the recombinant protein of Mycoplasma gallisepticum RRF.
[0015] A polyclonal antibody against Mycoplasma gallisepticum RRF protein was prepared by immunizing animals with the aforementioned recombinant Mycoplasma gallisepticum RRF protein.
[0016] Based on the RRF sequence of Mycoplasma gallisepticum published by NCBI, this invention analyzes its antigenicity, selects the full-length RRF sequence (183aa) as the antigen, optimizes the codons of the 183bp gene sequence, synthesizes the gene fragment, expresses pET32-RRF using a prokaryotic expression system, and prepares rabbit anti-TEX264 serum polyclonal antibodies by purifying and concentrating the recombinant RRF protein.
[0017] This invention fills the gap in the lack of commercially available anti-Mycoplasma gallisepticum RRF antibodies and provides basic data for the study of RRF in drug target screening.
[0018] The specific detection method for recombinant Mycoplasma gallisepticum RRF protein of the present invention has the following objectives:
[0019] 1) Ensure antibody specificity: Ensure that the polyclonal antibody against the recombinant Mycoplasma gallisepticum RRF protein can specifically recognize the RRF protein, and avoid non-specific binding or cross-reaction;
[0020] 2) Assess the suitability of the antibody: This antibody can be used for Western blotting;
[0021] 3) Verify the reliability of the antibody: Use Western blotting to verify the reliability and reproducibility of the antibody under different experimental conditions;
[0022] 4) Standardized testing procedures.
[0023] A method for preparing a polyclonal antibody against the above-mentioned Mycoplasma gallisepticum RRF protein specifically includes the following steps:
[0024] (1) Animals were immunized with the above-mentioned Mycoplasma gallisepticum RRF recombinant protein as an antigen;
[0025] (2) Obtain animal serum and purify it to obtain a polyclonal antibody against Mycoplasma gallisepticum RRF protein.
[0026] Furthermore, in step (1) above, the immunized animal is a rabbit.
[0027] Furthermore, in step (1) above, the immunization method is multiple injections on the back.
[0028] Furthermore, the aforementioned multiple injections on the back are specifically as follows: the first main injection uses Freund's complete adjuvant, and the second, third, and fourth immunizations use Freund's incomplete adjuvant for booster injections, all of which are fully mixed with an equal volume of antigen before injection.
[0029] Furthermore, in step (2) above, the purification method is as follows: the recombinant protein of Mycoplasma gallisepticum RRF is covalently linked to a Sepharose 4B column activated with cyanide bromide, 10 mL of antiserum is incubated with the affinity purification column overnight, HCl is used to pre-wash to remove impurities, glycine is used to elute the antibody, and PBS is used for neutralization and then dialyzed.
[0030] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:
[0031] The polyclonal antibody against Mycoplasma gallisepticum RRF of this invention can specifically recognize endogenous RRF protein in Mycoplasma gallisepticum and can be used in ELISA and Western Blot experiments, providing a material basis for studying RRF function. Attached Figure Description
[0032] Figure 1 This is a diagram showing the SDS-PAGE electrophoresis results of a large-scale expression of the pET32-RRF recombinant vector.
[0033] Lane 1 is the protein marker, lane 2 is the protein sample after elution with 20 mM imidazole once, lane 3 is the sample after elution with 20 mM imidazole twice, lane 4 is the sample after elution with 20 mM imidazole three times, lane 5 is the sample after elution with 300 mM imidazole once, and lane 6 is the sample after elution with 300 mM imidazole twice.
[0034] Figure 2 The image shows the Western blot results of RRF protein detection in Mycoplasma gallisepticum using RRF polyclonal antibody. Lanes 1-4 show the RRF protein expression results after treatment with RRF-specific drugs, and lane 5 shows the expression results after 10...9 The expression of RRF protein in Mycoplasma gallisepticum at CCU / mL bacterial count. Lane 6 is the positive control group. The expression of RRF protein in Mycoplasma gallisepticum after treatment. Detailed Implementation
[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0036] Example 1 Antigen Analysis
[0037] The full-length RRF sequence of Mycoplasma gallisepticum (WP_011113319) from NCBI was analyzed for protein secondary structure, hydrophilicity / hydrophobicity, and antigenicity. The full-length protein was selected as the immunogen. The amino acid sequence corresponding to the Mycoplasma gallisepticum RRF protein is shown in SEQ ID NO:1.
[0038] SEQ ID NO:1
[0039] MEFKTYSDFFDKNANSIINWFEGELAKVRSGRANLKILDNVRAEYYGEQTPLIEMASLSIPEPREILIKPYEKSSVNLIQAALLKANLNLTPVVDGDKIRIKLPLLTEENRKENVKKVKAVGEKAKQEVRFIRRDTLNKIKSDKIADKDLNKYFEEQVDKITKKYIDQIDSILAKKEKDLLSL.
[0040] The three-letter amino acid sequence corresponding to the Mycoplasma gallisepticum RRF protein is shown in SEQ ID NO:2.
[0041] SEQ ID NO:2
[0042] Met-Glu-Phe-Lys-Thr-Tyr-Ser-Asp-Phe-Phe-Asp-Lys-Asn-Ala-Asn-Ser-Ile-Ile-Asn-Trp-Phe-Glu-Gly -Glu-Leu-Ala-Lys-Val-Arg-Ser-Gly-Arg-Ala-Asn-Leu-Lys-Ile-Leu-Asp-Asn-Val-Arg-Ala-Glu-Tyr-Tyr -Gly-Glu-Gln-Thr-Pro-Leu-Ile-Glu-Met-Ala-Ser-Leu-Ser-Ile-Pro-Glu-Pro-Arg-Glu-Ile-Leu-Ile-Ly s-Pro-Tyr-Glu-Lys-Ser-Ser-Val-Asn-Leu-Ile-Gln-Ala-Ala-Leu-Leu-Lys-Ala-Asn-Leu-Asn-Leu-Thr-Pr o-Val-Val-Asp-Gly-Asp-Lys-Ile-Arg-Ile-Lys-Leu-Pro-Leu-Leu-Thr-Glu-Glu-Asn-Arg-Lys-Glu-Asn-V al-Lys-Lys-Val-Lys-Ala-Val-Gly-Glu-Lys-Ala-Lys-Gln-Glu-Val-Arg-Phe-Ile-Arg-Arg-Asp-Thr-Leu-A sn-Lys-Ile-Lys-Ser-Asp-Lys-Ile-Ala-Asp-Lys-Asp-Leu-Asn-Lys-Tyr-Phe-Glu-Glu-Gln-Val-Asp-Lys- Ile-Thr-Lys-Lys-Tyr-Ile-Asp-Gln-Ile-Asp-Ser-Ile-Leu-Ala-Lys-Lys-Glu-Lys-Asp-Leu-Leu-Ser-Leu.
[0043] To improve protein expression levels in prokaryotes, codon optimization was performed on the full-length nucleotide sequence of the RRF, as shown in SEQ ID NO:3, and gene fragments were synthesized.
[0044] SEQ ID NO:3
[0045] .
[0046] Example 2: Construction of pET32-RRF recombinant vector and preparation of engineered bacteria
[0047] The RRF gene fragment that was correctly synthesized and determined was directionally cloned into the pET-32 expression vector using T4 ligase to construct the recombinant plasmid pET32-RRF, and positive clones were identified.
[0048] After successful identification, the constructed plasmid was transformed into BL21 DE3 competent cells and inoculated onto resistant LB agar plates for overnight growth. Six single clones from the transformation plates were selected and inoculated with 60 μL of the bacterial culture into 200 mL of resistant medium, and cultured overnight at 37°C and 220 RPM. The next day, fresh resistant medium was added to 800 mL, and the cells were cultured for 1-2 h until the OD600nm value reached 0.5-0.6. 200 μL of 1 M IPTG (28°C or 37°C) was added to induce expression for 3.5 h. The cells were collected by centrifugation at 4°C (66 rpm × 15 min), the supernatant was discarded, 30 mL of PBST was added to suspend the cells, and 1 mM PMSF was added to the final concentration. The cells were then sonicated at 200 W for 6 min on ice. The cells were incubated on a shaker at 4°C for 1 h. The cells were centrifuged at 4°C at 133 rpm × 15 min, the supernatant was collected, and 400 μL of nickel column was added for binding overnight at 4°C. Collect the nickel column (33 rpm × 5 min), wash the beads with 20 mM imidazole washing buffer to remove contaminating proteins (1 mL × 3 times). Add 300 μL of 300 mM imidazole elution buffer, allow the elution buffer to fully bind with the beads for 1 h, centrifuge and collect the supernatant. Add another 300 μL of elution buffer to the beads, elute for 1 h, centrifuge and collect the supernatant, combine the two elution buffers into one tube. Dialyze to PBS buffer to change the buffer. SDS-PAGE is used to identify the protein molecular weight, purity and concentration. SDS-PAGE results are shown below. Figure 1 As shown.
[0049] Depend on Figure 1 It was found that after elution three times with 20 mM imidazole, the protein concentration was higher and the bands were more uniform. The RRF protein with the trx tag was a soluble supernatant with a molecular weight of 39 kDa. It did not degrade during expression and could be used for immunization.
[0050] Example 3: Preparation of polyclonal antibodies against Mycoplasma gallisepticum RRF protein
[0051] Initial immunization: 2-3 month old Japanese White rabbits were selected. 400 µg / 400 μL of the recombinant protein prepared in Example 2 was used as the immunogen to prepare Freund's complete adjuvant. Immunization was performed by subcutaneous injection into the rabbit's back.
[0052] The immunization cycle was 64 days, with a total of 5 immunizations. Booster immunizations were administered on days 28, 42, 47, and 54 after the initial immunization. The immunization dose was 150 µg / 150 μL. Final bloodletting and collection of antiserum were performed ten days after the fourth immunization.
[0053] Example 4: Antiserum purification
[0054] An affinity purification column was prepared by covalently linking 1 mg of purified protein to a hydrogen bromide-activated Sepharose 4B column using standard methods. 10 mL of antiserum was incubated overnight with the affinity purification column. The column was pre-washed with pH 5.0 HCl to remove impurities. Elution was then performed with pH 2.5 0.15 Mglycine buffer, followed by rapid neutralization with 10x PBS buffer to prepare the affinity-purified antibody. The PBS buffer was dialyzed to replace the buffer. The concentration of the purified antibody was determined using the Bradford method. The titers of the antiserum and affinity-purified antibody were detected by indirect ELISA. The ELISA titer results of the affinity-purified antibody are shown in Table 1.
[0055] Table 1. Results of Affinity-Purified Antibody ELISA Titer Assay
[0056] Grouping Antibody 1 Antibody 2 Antiserum 1 Antiserum 2 CK 0.072 0.068 0.07 0.068 2K 2.535 2.327 2.243 2.198 4K 2.067 1.844 1.749 1.602 8K 1.445 1.212 1.289 1.118 16K 0.891 0.758 0.766 0.522 32K 0.502 0.528 0.426 0.361 64K 0.27 0.326 0.289 0.194 128K 0.251 0.279 0.225 0.203
[0057] As shown in Table 1, the titer of the purified antibody (polyclonal antibody) is approximately 166, and the titer of the antiserum is approximately 8K. This indicates that the present invention successfully prepared a polyclonal antibody against Mycoplasma gallisepticum RRF protein, and the purified antibody has a higher titer.
[0058] Example 5: Specific detection of anti-Mycoplasma gallisepticum RRF polyclonal antibody immunoblotting
[0059] Protein sample preparation: Take 5 mL of 1×10 9 CCU / mL Mycoplasma gallisepticum culture was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and 1 mL of protein lysis buffer and 10 μL of protease phosphatase inhibitor were added. After sonication for 30 min, the supernatant was collected by centrifugation at 12000 rpm for 10 min, and the protein concentration was determined by the Bradford method. 5× denaturing protein loading buffer was added, and the mixture was heated in a metal bath at 95°C for 10 min.
[0060] Protein samples were separated by 10% SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane under constant current of 200 mA for 60 min. After transfer, the membrane was blocked with 0.5% skim milk powder for 2 h. The prepared polyclonal antibody against Mycoplasma gallisepticum RRF protein was diluted 1:0000 in the skim milk powder and incubated at 4°C for 12 h. The membrane was washed three times with PBST for 10 min each time. Goat anti-rabbit secondary antibody was added, and the membrane was incubated at room temperature for 1 h. After washing three times with PBST for 10 min each time, ultrasensitive ECL luminescent solution was added to the PVDF membrane surface for exposure. The Western blot results are as follows: Figure 2 As shown.
[0061] Depend on Figure 2It can be seen that, using the recombinant RRF protein sample of the present invention as the primary antibody, a specific band of Mycoplasma gallisepticum was detected at 24 kDa. This indicates that the polyclonal antibody against Mycoplasma gallisepticum RRF protein provided by the present invention can specifically recognize the Mycoplasma gallisepticum RRF protein.
[0062] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A recombinant protein of Mycoplasma gallisepticum RRF, characterized in that, The amino acid sequence is shown in SEQ ID NO:
1.
2. The recombinant Mycoplasma gallisepticum RRF protein according to claim 1, characterized in that, The three-letter amino acid sequence is shown in SEQ ID NO:2, and the full-length sequence is shown in SEQ ID NO:
3.
3. A recombinant Mycoplasma gallisepticum RRF protein according to claim 1 or 2, characterized in that, The plasmid used to construct the recombinant protein vector was the pET-32a+ expression vector.
4. A method for preparing the recombinant Mycoplasma gallisepticum RRF protein as described in claim 1, characterized in that, Specifically, the following steps are included: (1) Transform the recombinant protein vector into competent cells to construct engineered bacteria with the recombinant vector; (2) The engineered bacteria were induced by IPTG to express the recombinant protein of Mycoplasma gallisepticum RRF and then purified to obtain the recombinant protein of Mycoplasma gallisepticum RRF.
5. A polyclonal antibody against Mycoplasma gallisepticum RRF protein, characterized in that, It was prepared by immunizing animals with the recombinant Mycoplasma gallisepticum RRF protein as described in claim 1.
6. A method for preparing a polyclonal antibody against Mycoplasma gallisepticum RRF protein as described in claim 5, characterized in that, Specifically, the following steps are included: (1) Using the recombinant Mycoplasma gallisepticum RRF protein as described in claim 1 as an antigen to immunize animals; (2) Obtain animal serum and purify it to obtain the polyclonal antibody against Mycoplasma gallisepticum RRF protein.
7. The method for preparing a polyclonal antibody against Mycoplasma gallisepticum RRF protein according to claim 6, characterized in that, In step (1), the immunized animal is a rabbit.
8. The method for preparing a polyclonal antibody against Mycoplasma gallisepticum RRF protein according to claim 6, characterized in that, In step (1), the immunization method is multi-point injection on the back.
9. A method for preparing a polyclonal antibody against Mycoplasma gallisepticum RRF protein according to claim 8, characterized in that, The specific method of the back multi-point injection is as follows: the first main injection uses Freund's complete adjuvant, and the second, third and fourth immunizations use Freund's incomplete adjuvant for booster injections, all of which are fully mixed with an equal volume of antigen before injection.
10. A method for preparing a polyclonal antibody against Mycoplasma gallisepticum RRF protein according to claim 6, characterized in that, In step (2), the purification method is as follows: covalently linking the recombinant protein of Mycoplasma gallisepticum RRF to a Sepharose 4B column activated with cyanogen bromide, incubating 10 mL of antiserum with the affinity purification column overnight, pre-washing with HCl to remove impurities, eluting antibodies with glycine, neutralizing with PBS, and then dialysis.