Recombinant crassostrea gigas complement C3 splitting fragment rCgC3dg as well as preparation method and application thereof

Abstract

The invention discloses a recombinant crassostrea gigas complement C3 cleavage fragment rCgC3dg as well as a preparation method and application thereof. The amino acid sequence of the rCgC3dg is shown as SEQ ID NO. 1; the preparation method comprises the following steps: carrying out PCR (Polymerase Chain Reaction) amplification on a Crassostrea gigas CgC3dg coding region fragment by using primers P1 and P2, wherein the DNA (Deoxyribose Nucleic Acid) sequence of the primer P1 is as shown in SEQ ID NO. 2, and the DNA sequence of the primer P2 is as shown in SEQ ID NO. 3; respectively carrying out BamHI and Xhol I enzyme digestion on a PCR amplification product and a pET-30a carrier, and then connecting and transforming through T4 ligase to construct a recombinant; transferring the recombinant into an escherichia coli Transsetta (DE3) expression strain, culturing the expression strain, adding IPTG (isopropyl-beta-d-thiogalactoside) to carry out induced expression on the recombinant protein, and then purifying and renaturating to obtain the recombinant rCgC3dg protein. The recombinant crassostrea gigas complement C3 split fragment rCgC3dg and rCgGSDME-N can be used for preparing a medicine for resisting vibrio splendidus and escherichia coli after being mixed.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and particularly relates to a recombinant complement C3 cleavage fragment r from the oyster shell. Cg C3dg, its preparation method and its application. Background Technology

[0002] The complement system is an important component of the innate immune system. Activation of the complement system can participate in the immune response and trigger a series of efficient and complex immune effects. After complement C3 is activated, it produces a variety of functional active fragments. When the active C3 fragments bind to various pathogens, the complement fragment receptors on the surface of immune cells recognize the pathogens labeled by the active C3 fragments and initiate the phagocytosis-lysosome process to engulf and clear the pathogens.

[0003] Chinese invention patent number 202111324007.5 discloses a "recombinant oyster pyroporin protein r". Cg "GSDME-N, Preparation Method and Application" specifically discloses recombinant long oyster pyroporin protein r Cg GSDME-N can directly kill Vibrio splendidus, Escherichia coli, Staphylococcus aureus, and Micrococcus luteus, and can inhibit the growth of Vibrio splendidus and Staphylococcus aureus.

[0004] To date, no complement C3 cleavage fragments have been observed in the Pacific oyster. Cg C3dg can bind to Vibrio splenium and Escherichia coli, and can promote the development of pyroporin in oysters. Cg Reports on the direct bactericidal activity of GSDME-N against Vibrio brilliance and Escherichia coli. Summary of the Invention

[0005] This invention aims to address the aforementioned problems in the prior art by providing a recombinant oyster complement C3 cleavage fragment r. Cg C3dg, its preparation method and its application.

[0006] The technical solution of this invention is: a recombinant oyster complement C3 cleavage fragment r Cg C3dg, the amino acid sequence is shown in SEQ ID NO. 1.

[0007] A recombinant oyster complement C3 cleavage fragment r Cg The preparation method of C3dg is carried out in the following steps: a. Using primers P1 and P2 on the Pacific oyster. Cg The C3dg coding region fragment was amplified by PCR. The DNA sequence of primer P1 is shown in SEQ ID NO. 2, and the DNA sequence of primer P2 is shown in SEQ ID NO. 3. b. The PCR amplification products and pET-30a vector were respectively subjected to... Bam HI and Xhol After digestion with enzyme I, the cells were ligated using T4 ligase, transformed, and recombinants were constructed. c. Transform the recombinant into Escherichia coli. Transetta The recombinant protein was cultured in (DE3) expression strains and induced to express by IPTG. After purification and refolding, the recombinant oyster complement C3 cleavage fragment r was obtained. Cg C3dg.

[0008] A recombinant oyster complement C3 cleavage fragment r Cg The application of C3dg is related to r Cg GSDME-N mixtures are used in the preparation of drugs against Vibrio splendidus or Escherichia coli.

[0009] The preferred recombinant oyster complement C3 cleavage fragment r Cg C3dg and r Cg GSDME-N, when mixed at the same concentration in a 1:1 volume ratio, is used in the preparation of drugs against Vibrio splendidus or Escherichia coli.

[0010] This invention uses specific primer pairs for the long oyster. Cg The C3dg coding region fragment was amplified by PCR, and the PCR amplification product was then mixed with the pET-30a vector. Bam HI and Xhol After digestion with enzyme I, ligation was performed using T4 ligase, followed by transformation to construct recombinants; the recombinants were then transformed into *E. coli*. Transetta In the (DE3) expression strain, the expression strain was then cultured and IPTG was added to induce the expression of the recombinant protein. The protein was then purified and refolded to obtain the recombinant oyster complement C3 cleavage fragment r. Cg C3dg. The recombinant oyster C3 cleavage fragment prepared. Cg C3dg can bind to Vibrio splenium and Escherichia coli, and can promote the production of pyroporin in oysters. Cg GSDME-N has direct bactericidal activity against Vibrio splenium and Escherichia coli, and can be used to prepare drugs against Vibrio splenium and Escherichia coli. Attached Figure Description

[0011] Figure 1 This is a recombinant complement C3 cleavage fragment r from an embodiment of the present invention. Cg C3dg and recombinant long oyster pyroporin r Cg The results of GSDME-N co-incubation with bacteria were analyzed and statistically analyzed.

[0012] Figure 2 This is a recombinant complement C3 cleavage fragment r from an embodiment of the present invention.Cg C3dg and recombinant long oyster pyroporin r Cg Electron micrograph of the bactericidal effect of GSDME-N co-incubation on Escherichia coli.

[0013] Figure 3 This is a recombinant complement C3 cleavage fragment r from an embodiment of the present invention. Cg C3dg and recombinant long oyster pyroporin r Cg Electron micrograph of the bactericidal effect of GSDME-N co-incubation on Vibrio brilliance.

[0014] Figure 4 This is a recombinant complement C3 cleavage fragment r from an embodiment of the present invention. Cg C3dg and recombinant long oyster pyroporin r Cg Statistical chart of bactericidal effect after co-incubation with GSDME-N. Detailed Implementation

[0015] The recombinant oyster complement C3 cleavage fragment r of the present invention Cg The preparation method of C3dg is carried out in the following steps: 1. Construction of recombinant vectors The recombinant vector used in this embodiment of the invention is the Novagen pET-30a(+) prokaryotic expression vector. Using PCR technology, the 5' end was laced with... Bam HI and Xhol I. Restriction enzyme sites primers P1 and P2, for oyster shells Cg The C3dgmRNA coding region was amplified, and the DNA sequence of primer P1 is shown in SEQ ID NO. 2, and the DNA sequence of primer P2 is shown in SEQ ID NO. 3.

[0016] The PCR reaction conditions were as follows: first, pre-denaturation at 94℃ for 5 min, followed by the following cycles: denaturation at 94℃ for 30 s, annealing at 54℃ for 30 s, extension at 72℃ for 2 min, for a total of 30 cycles, and a final extension at 72℃ for 10 min; the amplified fragment was purified and recovered by agarose gel electrophoresis and ligated into the pMD19-T vector; after transformation, positive clones were screened, plasmids were extracted, and... Bam HI and Xhol I. The plasmid was double-digested with enzymes; the target fragment and the enzyme were recovered. Bam HI and Xhol The expression vector pET-30a(+) digested with enzyme I was ligated to complete the construction of the recombinant plasmid.

[0017] 2. Recombinant oyster complement C3 cleavage fragment r Cg C3dg expression The constructed recombinant plasmid was transformed and expressed in *E. coli* Transetta (DE3). Single colonies were picked and inoculated into 200 mL LB liquid medium and cultured at 220 rpm and 37°C until OD500. 600 =0.4-0.8; Add IPTG (final concentration 1 mmol / L), continue culturing for 4 h, then centrifuge at 10,000 rpm for 5 min at 4℃, collect the bacterial cells, and freeze at -80℃ for later use; at the same time, take 1 mL of bacterial culture, centrifuge, discard the supernatant, add 80 μL of water and 20 μL of 5× protein loading buffer, boil at 99℃ for 10 min, centrifuge briefly, and detect the expression product by SDS-PAGE.

[0018] 3. Recombinant oyster complement C3 cleavage fragment r Cg Purification and refolding of C3dg The expression product was purified using a nickel agarose gel FF column to obtain the denatured recombinant protein, which was then refolded by dialyzing with dialysis buffer. The specific operational steps are as follows: (1) Nickel agarose gel FF packing, 1.6×20 cm, column bed volume is 10 mL; (2) Equilibrate 2-5 bed volumes with buffer I (50 mmol / L Tris-HCl buffer, pH 9.0, 50 mmol / L NaCl, 8 mol / L urea) at a flow rate of 2 mL / min; (3) Take the cells that have been induced to express by IPTG, resuspend them in buffer I, sonicate them at 150 W for 30 min, centrifuge at 12,000 rpm and 4℃ for 30 min, filter the supernatant through a 0.45 μm filter membrane, and pass it through a column at a flow rate of 1 mL / min. (4) Wash with buffer I for 2-5 bed volumes at a flow rate of 2 mL / min; (5) Wash with 2-5 column volumes of 50 mmol / L imidazole buffer I at a flow rate of 2 mL / min; (6) Elute the target protein with imidazole buffer I containing 400 mmol / L and collect it; (7) Detect the expression of the fusion protein using SDS-PAGE; (8) Wash with pure water for 5 column volumes, then wash with 20% ethanol for 3 column volumes at a flow rate of 2 mL / min. Store the column at 4°C. Purifying recombinant protein in denature requires dialysis in a refolding buffer to remove urea, allowing the protein to refold correctly and restore its proper conformation. The denatured purified product was refolded by dialysis with 2 mM reduced glutathione, 0.4 mM oxidized glutathione, 1 mM EDTA, 50 mM Tris-HCl, 100 mM NaCl, 10% glycerol, 1% glycine, and a gradient of decreasing urea concentrations. The urea concentration was initially 6 M, gradually replaced with 4 M, 3 M, 2 M, 1 M, and 0 M. No glycerol was added during the final dialysis until no urea remained in the dialysate. Dialysis was performed at 4°C for 12 h each time to obtain the recombinant oyster complement C3 cleavage protein r. Cg C3dg, the obtained recombinant oyster complement C3 cleavage fragment r Cg The amino acid sequence of C3dg is shown in SEQ ID NO. 1.

[0019] Length: 299 amino acids Type: Amino acid Chain type: single chain Characteristics: Molecular weight is 34.21 kDa, and isoelectric point is 8.83.

[0020] Experimental Example 1: Recombinant complement C3 cleavage fragment r from the present invention (Example of the present invention) Cg C3dg binding activity assay The bacterial strains used in this experiment were obtained from the following sources: Vibrio brilliance ( Vibrio splendidus Purchased from Beijing Microbial Culture Collection Center, Escherichia coli (E. coli) Escherichia coli Purchased from Beijing Quanshijin Company.

[0021] The specific steps are as follows: (1) The above two microorganisms were cultured overnight. The culture method was as follows: Escherichia coli was cultured in LB medium at 37°C for 20 h, and Vibrio splenti was cultured in 2216E medium at 28°C for 20 h. (2) Collect bacterial cells by centrifugation, resuspend in TBS buffer, and adjust the bacterial concentration to 1×10⁻⁶. 8 CFU / mL; (3) Take 100 μL of the microbial suspension and mix it with an equal volume and concentration of the r obtained in the embodiments of the present invention. Cg C3dg, r Cg GSDME-N and r Cg GSDME-N+r Cg The mixture was incubated with C3dg (equal concentration, volume ratio 1:1) and then rotated at room temperature for 3 h. (4) Centrifuge at 4,000 rpm for 5 min to collect bacterial cells, and wash the bacterial cells four times with TBS buffer; (5) After washing, collect the bacterial cells and resuspend them in 40 μL of sterile water; (6) Add 10 μL of 5× protein electrophoresis buffer, heat at 99℃ for 10 min, and separate the protein samples by SDS-PAGE electrophoresis; (7) After electrophoresis, remove the gel and cut out NC membranes and filter paper of the same size and immerse them together in the electrophoresis buffer for 10 min. (8) Place the filter paper, NC membrane, gel and filter paper into the electroporation apparatus in order from top to bottom. Set the corresponding current according to the size of the gel block area and transfer the membrane for 45 minutes. (9) Remove the NC membrane and wash it three times with TBS buffer, 5 min each time; (10) Wash three times with TBST buffer, 5 min each time; (11) Place the NC membrane in 5% skim milk powder (dissolved in TBST) and seal at room temperature for 2 h; (12) Remove the NC membrane and wash it three times with TBST buffer, 5 min each time; (13) Immerse the NC membrane in a His-tagged monoclonal antibody (purchased from Shanghai Sangon Biotech) solution (5% skim milk powder, TBST buffer) diluted according to the ratio and incubate at 4°C for 16 h; (14) Remove the NC membrane and wash it three times with TBST buffer, 5 min each time; (15) Place the NC membrane in a solution of goat anti-mouse HRP secondary antibody (purchased from Shanghai Sangon Biotech) diluted according to the ratio (5% skim milk powder TBST buffer) and incubate at room temperature for 1 h; (16) Remove the NC membrane and wash it three times with TBST buffer, 5 min each time; (17) ECL method development, imaging recorder recording of western blotting results and statistical analysis, such as Figure 1 As shown.

[0022] The results show that the embodiment r of the present invention Cg C3dg, r Cg GSDME-N and r Cg GSDME-N+r Cg The C3dg mixture exhibited binding activity against both *Escherichia coli* and *Vibrio splendidus*. Statistical analysis showed that r Cg C3dg+r Cg r in GSDME-N mixture group Cg GSDME-N's binding activity against Escherichia coli and Vibrio splenida is related to r CgThe level was significantly higher in the GSDME-N group compared to the r group; Cg C3dg+r Cg r in GSDME-N mixture group Cg C3dg's binding activity against Escherichia coli and Vibrio splenida is related to r Cg There was no significant change compared to the C3dg group.

[0023] Experimental Example 2: Recombinant complement C3 cleavage fragment r from the present invention (Example of the present invention) Cg C3dg and pyroporin r from Oyster shell Cg The bactericidal effect of GSDME-N co-incubation on Escherichia coli r Cg C3dg and r Cg After co-incubation with GSDME-N (equal concentration, 1:1 volume ratio), it was incubated with E. coli, and then observed under an electron microscope. Cg C3dg and r Cg bactericidal activity after co-incubation with GSDME-N.

[0024] The strains were sourced as in Experimental Example 1.

[0025] The specific steps are as follows: (1) The experimental bacteria were prepared with a TBS-free solution to a concentration of approximately 4 × 10⁻⁶. 4 Bacterial suspension with cell / mL; (2) Take 100 μL of the microbial suspension and mix it with an equal volume and concentration of the r obtained in the invention example. Cg GSDME-N, r Cg C3dg, r Cg GSDME-N and r Cg The mixture of C3dg (equal concentration, volume ratio 1:1) and negative control group (rTRX) was incubated at room temperature for 3 h by rotation. (3) After incubation for 3 h, the cells were collected by centrifugation at 4,000 rpm for 5 min and washed four times with TBS buffer. (4) Fix the bacterial cells with 2.5% glutaraldehyde solution and incubate overnight at 4°C; (5) After centrifugation, the sample was washed three times with TBS solution to remove glutaraldehyde. The sample was then dehydrated using a stepwise ethanol dehydration method, with ethanol concentrations of 30%, 50%, 70%, 80%, 90%, and 100%. Each step was treated for 15 minutes, followed by centrifugation, discarding the supernatant, and adding the next stage of ethanol. This process continued until 100% ethanol was added. The sample was then evenly spread onto a glass slide, dried at room temperature, and then sputtered with gold using an ion sputtering instrument before being observed under a scanning electron microscope. The results are as follows: Figure 2 As shown.

[0026] The results show that r CgGSDME-N, r Cg GSDME-N and the embodiments of the present invention are recombined. Cg The C3dg mixture exhibited puncture-killing effects against Escherichia coli, but r Cg C3dg+r Cg The GSDME-N mixture showed a more significant killing effect on perforated E. coli, while r Cg C3dg and rTRX had almost no perforation-killing effect on E. coli. Statistical results are as follows: Figure 4 As shown in the middle left figure.

[0027] Experimental Example 3: The long oyster r of the embodiment of the present invention Cg C3dg and r Cg Electron microscopy results of GSDME-N co-incubation for killing Vibrio strobilus r Cg C3dg+r Cg After co-incubation with GSDME-N (equal concentration, 1:1 volume ratio), the oyster was incubated with *Brachys brevicornu*, and then observed under an electron microscope. Cg C3dg+r Cg bactericidal activity after co-incubation with GSDME-N.

[0028] The strains were sourced as in Experimental Example 1.

[0029] The specific steps are as follows: (1) The experimental bacteria were prepared with a TBS-free solution to a concentration of approximately 4 × 10⁻⁶. 4 Bacterial suspension with cell / mL; (2) Take 100 μL of the microbial suspension and mix it with an equal volume and concentration of the r obtained in the invention example. Cg GSDME-N, r Cg C3dg, r Cg GSDME-N and r Cg The mixture of C3dg (equal concentration, volume ratio 1:1) and negative control group (rTRX) was incubated at room temperature for 3 h by rotation. (3) After incubation for 3 h, the cells were collected by centrifugation at 4,000 rpm for 5 min and washed four times with TBS buffer. (4) Fix the bacterial cells with 2.5% glutaraldehyde solution and incubate overnight at 4°C; (5) After centrifugation, the sample was washed three times with TBS solution to remove glutaraldehyde. The sample was then dehydrated using a stepwise ethanol dehydration method, with ethanol concentrations of 30%, 50%, 70%, 80%, 90%, and 100%. Each step was treated for 15 minutes, followed by centrifugation, discarding the supernatant, and adding the next stage of ethanol. This process continued until 100% ethanol was added. The sample was then evenly spread onto a glass slide, dried at room temperature, and then sputtered with gold using an ion sputtering instrument before being observed under a scanning electron microscope. The results are as follows: Figure 3 As shown.

[0030] The results show that the embodiment r of the present invention Cg GSDME-N and r Cg GSDME-N+r Cg The C3dg mixture exhibits a puncture-killing effect against Vibrio brilliance, but r Cg GSDME-N+r Cg The C3dg mixture showed a more significant killing effect on Vibrio brilliance pores, while r Cg C3dg and rTRX showed no effect on killing Vibrio splenita through perforation. Statistical results are as follows: Figure 4 As shown in the middle right figure.

Claims

1. A recombinant complement C3 cleavage fragment r from oyster shell Cg C3dg, characterized by: The amino acid sequence characteristics are shown in SEQ ID NO.

1.

2. A recombinant oyster complement C3 cleavage fragment r as described in claim 1 Cg The method for preparing C3dg is characterized by: Follow these steps in sequence: a. Using primers P1 and P2 on the Pacific oyster. Cg The C3dg coding region fragment was amplified by PCR. The DNA sequence of primer P1 is shown in SEQ ID NO. 2, and the DNA sequence of primer P2 is shown in SEQ ID NO.

3. b. The PCR amplification products and pET-30a vector were respectively subjected to... Bam HI and Xhol After digestion with enzyme I, the cells were ligated using T4 ligase, transformed, and recombinants were constructed. c. Transform the recombinant into Escherichia coli. Transetta The recombinant protein was cultured in (DE3) expression strains and induced to express by IPTG. After purification and refolding, the recombinant oyster complement C3 cleavage fragment r was obtained. Cg C3dg.

3. A recombinant oyster complement C3 cleavage fragment r as described in claim 1 Cg The application of C3dg is characterized by: r Cg C3dg and r Cg GSDME-N mixtures are used in the preparation of drugs against Vibrio splendidus or Escherichia coli.

4. The recombinant oyster complement C3 cleavage fragment r according to claim 4 Cg The application of C3dg is characterized by: The recombinant long oyster complement C3 cleavage fragment r Cg C3dg and r Cg GSDME-N, when mixed at the same concentration in a 1:1 volume ratio, is used in the preparation of drugs against Vibrio splendidus or Escherichia coli.

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