CN107163148A - Recombinant protein of pulmonary surfactant protein B propetides and its preparation method and application - Google Patents

Abstract

The invention relates to the field of genetic engineering, and discloses a recombinant protein of pulmonary surfactant protein B propeptide, a preparation method and application thereof. Specifically, the recombinant protein has the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence with a tag attached to the amino terminus and/or carboxyl terminus of SEQ ID NO: 1. The invention adopts the method of genetic engineering to obtain the proSP-B recombinant protein, the proSP-B recombinant protein has low cytotoxicity, exhibits good antibacterial activity, and has good practical application prospect.

Description

Recombinant protein of pulmonary surfactant protein B propeptide and its preparation method and application

technical field

The present invention relates to the field of genetic engineering, in particular to a recombinant protein of pulmonary surfactant protein B propeptide, a nucleic acid encoding the recombinant protein, a recombinant plasmid, a recombinant bacterial strain, and a method for preparing pulmonary surfactant protein B propeptide Methods of recombinant proteins and their use.

Background technique

Since the discovery of antibiotics, they have played an important role in the treatment of human diseases, cured many infectious diseases that humans were helpless, saved many lives, and greatly increased the average life expectancy of humans. However, due to their large-scale use, abuse and irregular use, a large number of drug-resistant strains appear and spread, which complicates the treatment of infectious diseases, increases treatment costs, increases morbidity and mortality, and makes the resistance of infectious bacteria Drug property has become an unavoidable problem. Moreover, these traditional anti-infective drugs also have many obvious side effects such as ototoxicity, nephrotoxicity, hepatotoxicity, neurotoxicity, cardiotoxicity, allergy and damage to the hematopoietic system, thus causing some unpredictable effects on patients. It may even endanger the life of the patient. Due to the many unfavorable factors of traditional antibiotics, it is of practical significance to find effective substitutes for antibiotics and develop new antibacterial drugs with low toxicity and good effect.

As a new class of antibacterial drugs, antimicrobial peptides have developed rapidly in recent years. Among them, pulmonary surfactant is a substance expressed and secreted by the body's own cells, and may have anti-inflammatory and antibacterial activities. Therefore, the development of a class of antibacterial drugs based on pulmonary surfactant has great prospects. Lung surfactant protein (surfactant protein, SP) is one of the most important substances in lung surfactant, especially the antibacterial effect of SP-B protein, which has attracted more and more attention. However, Marnie et al. found that the antibacterial effect of mature SP-B protein only occurs in vitro. In vivo, mature SP-B and phospholipids are associated with each other. The combination of phospholipids and SP-B is relatively stable, which cannot make SP-B It acts on foreign bacteria to exert its antibacterial function, and the mature SP-B acts on the phospholipid molecular layer non-selectively, lysing the cell membrane, showing non-selective lysing of somatic cells, especially red blood cells, causing body damage such as hemolysis. Therefore, people have turned their attention to prosurfactant protein-B (proSP-B), but proSP-B is difficult to obtain by chemical synthesis due to its strong hydrophobicity, which increases the The further development and research of this kind of substance has been carried out. Therefore, there is an urgent need to find a method to obtain proSP-B with good antibacterial activity, so as to lay the foundation for the development of new antibacterial drugs that can overcome many defects of traditional antibiotics and have good antibacterial effects.

Contents of the invention

The purpose of the present invention is in order to overcome the above-mentioned problem that prior art exists, a kind of recombinant protein of proSP-B is provided, the nucleic acid of coding this recombinant protein, recombinant plasmid, recombinant bacterial strain and the method for preparing the recombinant protein of proSP-B and their application. By adopting the method provided by the invention, the proSP-B recombinant protein is successfully obtained. The proSP-B recombinant protein has low cytotoxicity and exhibits good antibacterial activity.

In order to achieve the above object, in the first aspect, the present invention provides a recombinant protein of pulmonary surfactant protein B propeptide, wherein the recombinant protein has the amino acid sequence shown in SEQ ID NO: 1, or in SEQ ID NO: The amino-terminal and/or carboxy-terminal of 1 is linked with a tagged amino acid sequence.

In the second aspect, the present invention also provides a nucleic acid capable of encoding the above-mentioned recombinant protein.

In the third aspect, the present invention also provides a recombinant plasmid, which contains the above-mentioned nucleic acid.

In the fourth aspect, the present invention also provides a recombinant strain containing the above-mentioned recombinant plasmid.

In the fifth aspect, the present invention also provides a method for preparing a recombinant protein of pulmonary surfactant B propeptide, wherein the method comprises the following steps:

(1) transforming the above-mentioned recombinant plasmid into an expression host bacterium to obtain a recombinant strain;

(2) After the recombinant strain is cultured, it is induced and cultured in the presence of an inducer to obtain a culture solution;

(3) Purify and remove the label from the culture medium obtained in step (2) to obtain the recombinant protein of pulmonary surfactant B propeptide.

The above-mentioned recombinant protein, nucleic acid, recombinant plasmid, recombinant bacterial strain, and the recombinant protein prepared by the above-mentioned method are used in the preparation of antibacterial drugs; preferably, the antibacterial drugs are anti-Staphylococcus aureus, Escherichia coli ), Microsporum gypseum, Monilia albican, Trichophyton rubrum, Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus (Methicillin- resistantStaphylococcus aureus).

proSP-B is difficult to obtain by chemical synthesis due to its strong hydrophobicity. The present invention adopts genetic engineering methods, especially by using a specific expression vector (pGEX4T-1) and expression host bacteria (Escherichia coli BL21 (DE3) pLysS strain), successfully obtained proSP-B recombinant protein with higher concentration, and, by fusing the GST tag on the pGEX4T-1 vector with proSP-B, because the GST tag is hydrophilic, proSP-B can be significantly improved The water solubility of the recombinant protein expands its application range.

In addition, the proSP-B recombinant protein provided by the present invention exhibits good broad-spectrum antibacterial activity, and it has a strong inhibitory effect on common clinical bacteria and fungi, especially Staphylococcus aureus, Escherichia coli, gypsum-like Microsporum, Candida albicans, Trichophyton rubrum, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus showed significant inhibitory activity. Further research found that proSP-B recombinant protein may exert its antibacterial effect by affecting the permeability of bacterial cell membrane, thereby inhibiting the normal growth of bacteria. At the same time, the proSP-B recombinant protein provided by the present invention has less cytotoxicity, even if the protein concentration is increased to 1mg/mL (this concentration is far greater than its MIC value for bacteria), the proliferation ability of the cells is still higher than 59%, This shows that the proSP-B recombinant protein provided by the present invention has higher safety.

Description of drawings

What Fig. 1 shows is the result of PCR product identification in the embodiment of the present invention 1;

Figure 2 shows the identification results of the recombinant plasmid pET30-proSP-B in Example 1 of the present invention;

Figure 3 shows the identification results of the recombinant plasmid pGEX4T-1-proSP-B in Example 1 of the present invention;

Figure 4 shows the SDS-PAGE results of the induced expression of proSP-B recombinant protein in Example 1 of the present invention;

Figure 5 shows the Western-blot results of proSP-B recombinant protein induced expression in Example 1 of the present invention;

Figure 6 shows the purified SDS-PAGE and SDS-PAGE of the proSP-B recombinant protein induced and expressed in Example 1 of the present invention;

Fig. 7 shows the results of the antibacterial spectrum measurement of the proSP-B recombinant protein in Example 3 of the present invention.

detailed description

Neither the endpoints nor any values of the ranges disclosed herein are limited to such precise ranges or values, and these ranges or values are understood to include values approaching these ranges or values. For numerical ranges, between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, these values Ranges should be considered as specifically disclosed herein.

In a first aspect, the present invention provides a recombinant protein of pulmonary surfactant protein B propeptide, wherein the recombinant protein has the amino acid sequence shown in SEQ ID NO: 1, or at the amino terminal of SEQ ID NO: 1 and / or the amino acid sequence with a tag attached to the carboxy terminus.

The amino acid sequence shown in SEQ ID NO: 1 is: AGANDLCQECEDIVHLLTKMTKEDAFQDTIRKFLEQECDILPLKLLVPRCRQVLDVYLPLVIDYFQGQIKPKAICSHVGLC.

In the present invention, the "amino acid sequence linked with a tag" refers to the addition and modification of the amino acid sequence shown in SEQ ID NO: 1 using tags common in the art. Considering the strong hydrophobicity of proSP-B, the label is preferably hydrophilic. More preferably, the label is a GST label.

In the present invention, the GST (Glutathione S-transferase, glutathione S-transferase) tag can be a GST tag routinely used in the art, which can be introduced through an expression vector. In a preferred case, the GenBank number of the amino acid sequence of the GST tag is: AMQ34028.1 (see https://www.ncbi.nlm.nih.gov/protein/1005883217 for details ).

In the second aspect, the present invention also provides a nucleic acid capable of encoding the above-mentioned recombinant protein.

According to the present invention, the nucleic acid has the nucleic acid sequence shown in SEQ ID NO: 2, or a nucleic acid sequence with a tag attached to the amino terminus and/or carboxyl terminus of SEQ ID NO: 2; preferably, the tag has a hydrophilic Sex; more preferably, the label is a GST label.

SEQ ID NO：2所示的核酸序列为： gcaggagctaatgacctgtgccaagagtgtgaggatattgtccacctcctcacaaagatgaccaaggaagacgcttt ccaggacacgatccggaagttcctggaacaagaatgtgatatcctacccttgaagctgcttgtgccccggtgtcgcc aagtgcttgatgtctacctgcccctggttatcgactacttccagggccagattaaacccaaagccatctgcagtcatgt gggcctgtgc。

In the present invention, since the nucleic acid provided by the present invention has strong hydrophobicity, it is difficult to obtain by artificial synthesis. Therefore, the nucleic acid provided by the present invention is usually obtained by genetic engineering methods, for example, it can be obtained by polymerase chain reaction ( obtained by PCR amplification. Specifically, those skilled in the art can easily obtain templates and corresponding primers based on the nucleotide sequences provided by the present invention, and use PCR to amplify to obtain related sequences. Once the relevant nucleic acid sequence is obtained, the relevant amino acid sequence can be obtained in large quantities by recombinant methods. Usually, the obtained nucleic acid sequence is cloned into an expression vector, and then transformed into an expression host bacterium, and then the relevant nucleic acid sequence is obtained from the proliferated expression host bacterium by conventional methods.

In the present invention, the "nucleic acid sequence linked with a tag" refers to the addition and modification of the nucleic acid sequence shown in SEQ ID NO: 2 using common tags in the art. Considering the strong hydrophobicity of proSP-B, the tag is preferably hydrophilic. More preferably, the label is a GST label.

In the present invention, the GST tag can be a GST tag routinely used in the art, which can be introduced by an expression vector, preferably by a pGEX4T-1 plasmid. In a preferred case, the nucleic acid sequence of the GST tag is the sequence shown in the 258th to 959th positions of GenBank number: KU312308.1.

In the third aspect, the present invention also provides a recombinant plasmid, which contains the above-mentioned nucleic acid.

In the present invention, the plasmids used for the recombinant plasmids are preferably pET30a plasmids and/or pGEX4T-1 plasmids (both of which can be obtained commercially). The recombinant plasmid can be digested with various endonucleases capable of cutting sites at the multiple cloning site of the vector to obtain a linear plasmid, and then ligated with nucleic acid fragments cut with the same endonuclease to obtain a recombinant plasmid. For example, for pET30a plasmid, endonucleases such as Xhol I, Not I, Eag I, Hind III, EcoRI, BamH I, Nco I, Kpn I, BgI II, Nde I can be used; for pGEX4T-1 plasmid, Xhol can be used I, Not I, EcoR I, BamH I, Sma I and other endonucleases.

In a preferred embodiment of the present invention, the recombinant plasmid is an expression vector obtained by inserting the nucleic acid sequence shown in SEQ ID NO: 2 between the BamH I and Xho I restriction sites of the pGEX4T-1 plasmid .

In the fourth aspect, the present invention also provides a recombinant bacterial strain, wherein the recombinant bacterial strain contains the above-mentioned recombinant plasmid; preferably, the recombinant bacterial strain is Escherichia coli BL21(DE3) pLysS strain containing the above-mentioned recombinant plasmid

In the present invention, the recombinant plasmid can be transformed into the expression host bacterium by the conventional methods used in the field to obtain the recombinant strain. For example, heat shock method, calcium chloride chemical conversion method, etc. can be used, and heat shock method is preferable.

In the present invention, the expression host bacteria can be any suitable bacteria or fungi conventionally used in the art as hosts, for example, Escherichia coli. Preferably, the Escherichia coli DH5α strain used for preserving the recombinant plasmid, and the Escherichia coli BL21(DE3)pLysS strain used for expression. The expression host bacteria used in the present invention can be obtained through conventional commercial means.

In the fifth aspect, the present invention also provides a method for preparing a recombinant protein of pulmonary surfactant B propeptide, wherein the method comprises the following steps:

(1) transforming the above-mentioned recombinant plasmid into an expression host bacterium to obtain a recombinant strain;

(2) After the recombinant strain is cultured, it is induced and cultured in the presence of an inducer to obtain a culture solution;

(3) Purify and remove the label from the culture medium obtained in step (2) to obtain the recombinant protein of pulmonary surfactant B propeptide.

In the present invention, in step (1), the recombinant plasmid can be transformed into an expression host bacterium by a method commonly used in the art to obtain a recombinant strain. For example, heat shock method, calcium chloride chemical conversion method, etc. can be used, and heat shock method is preferable.

In the present invention, in step (1), the expression host bacteria can be any suitable bacteria or fungi conventionally used in the art as hosts, for example, it can be Escherichia coli. Preferably, the expression host bacterium used for preserving the recombinant plasmid is Escherichia coli DH5α strain, and the expression host bacterium used for expression is Escherichia coli BL21(DE3)pLysS strain. The expression host bacteria used in the present invention can be obtained through conventional commercial means.

In the present invention, in step (2), the present invention has no special limitation on the type of the inducer, which can be a conventional choice in the field, for example, the inducer can be isopropyl-β-d- Thiogalactoside (IPTG), and the concentration of the inducer is 0.5-2mmol/L, preferably 0.8-1.5mmol/L.

In the present invention, in step (2), the present invention has no special limitation on the conditions of the induction culture, as long as the purpose of inducing the expression of the recombinant protein can be achieved. In a preferred situation, the conditions for the induction culture include: the temperature is 28-37°C, preferably 28-35°C, more preferably 28-32°C; the time is more than 3h, preferably 3-24h, more preferably 12-24h.

In the present invention, in step (3), the present invention has no particular limitation on the purification and label removal methods, as long as the purpose of protein purification and label removal can be achieved, for example, protein purification can be carried out by adsorption. For purification, proteases can be used for tag removal. In a specific embodiment of the present invention, for the pGEX4T-1 plasmid, a GSTrap FF 16/10 prepacked column (obtainable through conventional commercial means) is used to purify the recombinant protein and remove the tag.

In the sixth aspect, the present invention also provides the above-mentioned recombinant protein, the above-mentioned nucleic acid, the above-mentioned recombinant plasmid, the above-mentioned recombinant strain, and the application of the recombinant protein prepared by the above-mentioned method in the preparation of antibacterial drugs; preferably, the antibacterial drug is anti-gold Staphylococcus aureus, Escherichia coli, Microsporum gypseum, Monilia albican, Trichophytonrubrum, Pseudomonas aeruginosa And the drug of at least one in methicillin-resistant Staphylococcus aureus (Methicillin-resistant Staphylococcus aureus); bacteria, trichophyton rubrum and methicillin-resistant staphylococcus aureus; further preferably, the antibacterial drug is anti-staphylococcus aureus and/or methicillin-resistant staphylococcus aureus .

In the application of the present invention, the pH value of the above-mentioned recombinant protein, the above-mentioned nucleic acid, the above-mentioned recombinant plasmid, the above-mentioned recombinant bacterial strain, and the recombinant protein prepared by the above-mentioned method used in the antibacterial drug is 3-8, preferably 5 -6, more preferably 5.4-6.

The present invention will be described in detail below by way of examples.

In the following examples:

SD rats were purchased from the Experimental Animal Center of the Field Surgery Institute of Daping Hospital;

Escherichia coli DH5α was purchased from Shanghai Angyu Biotechnology Co., Ltd.; Escherichia coli BL21(DE3) plysS was purchased from Beijing Huayueyang Biological Company. Bacteria and Pseudomonas aeruginosa were purchased from Beijing Beina Chuanglian Biotechnology Research Institute, and methicillin-resistant Staphylococcus aureus (No. ATCC 43300) was purchased from Guangdong Microbial Culture Collection Center;

CCL-149 cell line was purchased from Shanghai Guyan Biotechnology Co., Ltd.; pGEX4T-1 was purchased from Shanghai Youke Biotechnology Co., Ltd.; BamH I, Hind III, and Xho I were purchased from NEB Company in the United States; T4 ligase and total RNA extraction reagents All kits and reverse transcription kits were purchased from GeneCopoeia, USA; DNA gel recovery kit, DNA Loading Buffer, and DNAMarker were purchased from Takara, Japan; propidium iodide (PI) was purchased from Shanghai Xiangsheng Biotechnology Co., Ltd.; LDH kit was purchased from Biyuntian Biotechnology Co., Ltd. The remaining experimental reagents and consumables can be obtained through conventional commercial means.

Example 1

This example is used to illustrate the preparation method of proSP-B recombinant protein provided by the present invention.

1. Extraction of total RNA from rat lung tissue

(1) Total RNA was extracted from rat lung tissue using a total RNA extraction kit (purchased from GeneCopoeia, USA);

(2) Electrophoretic detection of RNA

Use a newly configured gel electrophoresis solution for electrophoresis, and a gel imager to detect and analyze the quality of RNA.

2. Acquisition and amplification of proSP-B gene cDNA

(1) Obtain cDNA fragments

Reverse transcription reaction system:

Total RNA 1 μg

Oli(dT) ₁₈ 1 μL

Free RNase Water to 13μL

React at 65°C for 10 min, then store at 4°C.

reaction system:

Reaction conditions: react at 42°C for 60 minutes, then react at 85°C for 5 minutes, and then store at 4°C. Finally, the reverse transcription product was stored in a -80°C refrigerator for a long time.

(2) PCR amplification using cDNA as template

(a) Primer sequence design

According to the pET30a plasmid map and the restriction sites of BamHI and HindIII, the upstream and downstream primers were designed with Primer Premier 5.0 software. The primer sequences are as follows.

proSP-B-F 5'-AAAGGATCCGCAGGAGCTAATGACCTG-3' (SEQ ID NO: 3)

proSP-B-R 5'-TTTAAGCTTTTAGCACAGGCCCACATG-3' (SEQ ID NO: 4)

(b) Reaction solution preparation:

(c) PCR reaction conditions: react at 95°C for 5 min; denature at 95°C for 30 s, anneal at 64°C for 30 s, and extend at 72°C for 45 s, a total of 39 cycles; extend at 72°C for 6 min, and store at 4°C.

(d) PCR product identification

After the PCR reaction, agarose gel electrophoresis was performed for detection, and the bands were observed on a gel imaging system to analyze the results. The results are shown in Figure 1. It can be seen from Figure 1 that a band appeared at about 250 bp, and the size conformed to the size of the target gene, which preliminarily proved that the reverse transcription was successful.

(e) Purify the PCR product with a PCR product purification and recovery kit.

(3) Construction and identification of pET30-proSP-B expression vector

The pET30 and proSP-B fragments were double digested with BamH I and Hind III, and the digested products were recovered by cutting the gel, and ligated overnight at 4°C with T4 DNA ligase.

(a) BamH I, Hind III double enzyme digestion reaction system:

Reaction conditions: constant temperature water bath, 37°C, 8h, agarose gel electrophoresis detection.

(b) Gel cutting and recovery of vectors and target fragments

The gel cutting recovery and purification of the product were carried out according to the instructions of the DNA purification and recovery kit.

(c) T4 DNA ligase reaction system:

(d) Transformation and identification of recombinant plasmids:

Add 10 μL of the ligation product to 100 μL of Escherichia coli DH5α competent, and use a PCR machine to carry out the plasmid introduction operation for transformation. The process is as follows: react at 0°C for 30 minutes, then react at 42°C for 30 seconds, and then store at 0°C.

Then, 300 μL of liquid LB medium was added to the product, and cultured at 37° C. and 150 r/min for 1 hour. Then spread it on a solid LB plate containing 50 μg/mL kana, culture overnight at 37°C, then pick colonies for colony PCR identification, and identify positive colonies for plasmid expansion, plasmid PCR and double enzyme digestion identification. The recombinant plasmid identified as positive was named pET30-proSP-B, and sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing. Sequencing results show that the 187-429 positions of the recombinant plasmid sequence are consistent with the sequence shown in SEQ ID NO:2.

The PCR results are shown in Figure 2. Specifically, the ligation product was transformed into DH5α competent cells, screened on a resistance plate containing kana, the PCR identification results of the colonies are shown in Figure 2 (a), and the results of agarose electrophoresis of the colony shake flask plasmids are shown in Figure 2 ( As shown in b), the PCR identification result of the colony plasmid is as shown in Figure 2 (c), and the result of the double enzyme digestion of the colony plasmid is as shown in Figure 2 (d), as can be seen from the figure, the PCR results are all single bands, and their size is about 250bp, consistent with the size of the target gene proSP-B, the double restriction map also shows a single band at 250bp, the position is correct, consistent with the size of proSP-B, preliminary proof that the recombinant plasmid pET30-proSP-B was successfully constructed.

Moreover, according to the results of BLAST in NCBI, the sequencing result of the positive plasmid matched proSP-B, which indicated that the pET30-proSP-B vector constructed during the experiment was successfully constructed.

(4) Construction and identification of pGEX4T-1-proSP-B expression vector

The pGEX4T-1 and pET30-proSP-B plasmids were digested with BamH I and Xho I, and the digested products were recovered by gel cutting, and ligated overnight at 4°C with T4 DNA ligase.

(a) Enzyme digestion reaction system:

Double enzyme digestion reaction conditions: constant temperature water bath, 37°C, 8h, agarose gel electrophoresis detection.

(b) T4 DNA ligase reaction system:

(c) Transformation and identification of recombinant plasmids:

The ligation product was transformed into Escherichia coli DH5α competent, and colony PCR identification was carried out. The identified positive colonies were expanded to extract plasmids, plasmid PCR and double enzyme digestion identification. The recombinant plasmid identified as positive was named pGEX4T-1-proSP-B, and sent to Sangon Bioengineering (Shanghai) Co., Ltd. for sequencing. Sequencing results show that the 609-851 positions of the recombinant plasmid sequence are consistent with the sequence shown in SEQ ID NO: 2.

The PCR results are shown in Figure 3. Specifically, the ligation product was transformed into DH5α competent cells, and screened on a resistance plate containing ampicillin. The PCR identification results of the colonies were shown in Figure 3(a), and the results of agarose electrophoresis of the colony shake flask plasmids were shown in Figure 3(b) ), the results of PCR identification of colony plasmids are shown in Figure 3(c), and the results of double enzyme digestion of colony plasmids are shown in Figure 3(d). It can be seen from the figure that the PCR results are all single bands, and their size is about 250bp , which is consistent with the size of the target gene proSP-B, and the double restriction map also shows a single band at 250bp, which is in the correct position and consistent with the size of proSP-B, which preliminarily proves that the recombinant plasmid pGEX4T-1-proSP-B was successfully constructed.

Moreover, according to the results of BLAST in NCBI, the sequencing result of the positive plasmid matched proSP-B, which indicated that the pGEX4T-1-proSP-B vector constructed during the experiment was successfully constructed.

(5) ProSP-B recombinant protein induced expression and SDS-PAGE detection

The sequencing results showed that the correct recombinant plasmid pGEX4T-1-proSP-B was transformed into Escherichia coli BL21(DE3)plysS. Cultivate overnight at 37°C and 250r/min, transfer to fresh LB liquid medium containing Amp 50μg/mL the next day at an inoculum size of 1:100, culture with shaking at 37°C and 250r/min, when the OD600 of the bacterial solution is about At 0.8, draw part of the bacterial solution as a control before induction, and collect it by centrifugation at 12000r/min; add IPTG to the bacterial solution to induce a final concentration of 1.0mmol/L. , 6h, 9h, 12h, and 24h to collect the bacteria, and use SDS-PAGE to detect and analyze.

SDS-PAGE dispensing system:

SDS-PAGE conditions:

Electrophoresis was carried out at a constant voltage, in which the concentration gel was 80V, and the separation gel was 120V. The electrophoresis was stopped when the protein markers were completely separated and occupied 3/4 of the single-line gel.

Staining: remove the stacking gel from the gel after electrophoresis, place the separating gel in Coomassie Brilliant Blue R250 staining solution, and stain on a horizontal shaker at room temperature for 1 hour; then decolorize with decolorization solution to completely remove the blue part of Coomassie Brilliant Blue R250 on the gel Clean, take out, and take pictures.

The result is shown in 4. Specifically, Figure 4(A) shows the results of SDS-PAGE gel running after induction at 37°C and 30°C, respectively. Among them, band 1 shows the protein expression results of uninduced bacteria; band 2 shows the protein expression results of bacteria induced at 37°C; band 3 shows the protein expression results of bacteria induced at 30°C The protein expression result of the bacterium; band 4 shows the protein expression result of the broken supernatant of the bacteria at 30°C; band 5 is the protein marker. It can be seen from Figure 4(A) that the amount of protein induced under the condition of 37°C is not as large as that induced under the condition of 30°C, and the amount of protein contained in the cell supernatant is basically the same as that of the whole cell, which shows that At 30°C, the recombinant protein exists in the form of soluble protein, and it is more appropriate to induce the protein at 30°C.

Figure 4(B) shows the protein expression after induction at 30°C for different time periods. Among them, band 1 is the protein marker; band 2 shows the protein expression result of the uninduced cells; band 3 shows the protein expression result of the bacterial cell supernatant after induction for 3 h; band 4 shows the The protein expression result of the bacterial supernatant after 6 hours of induction; the band 5 shows the protein expression result of the bacterial supernatant after the induction of 9 hours; the band 6 shows the protein expression result of the bacterial supernatant after the induction of 12 hours; the band 7 shows the protein expression result of the bacterial supernatant after induction for 24 hours. It can be seen from Figure 4(B) that under the condition of 30°C, after induction for 3-24 hours, the expression level of the target protein gradually increased with time, and the increase trend slowed down after 12 hours.

(6) Western Blot detection of proSP-B expression

After the protein was separated by SDA-PAGE, the region where GST-proSP-B was located was cut out according to the position of the protein marker, transferred to PVDF membrane, and electroporated at a constant current, 250mA, for 1h. Block with TBS-T containing 5% skimmed milk powder for 1 h; incubate with rabbit anti-rat proSP-B polyclonal antibody (diluted 1:10000) overnight at 4°C; wash with TBS-T the next day, and label with HRP Goat anti-rabbit IgG secondary antibody (diluted 1:5000) was incubated at room temperature for 1 h, then washed with TBS-T, and Chemiluminescent HRP Substrate luminescent solution was added dropwise, and imaged with Tianneng Luminescent Imaging Workstation.

The results are shown in Figure 5, in which band 1 shows the protein expression results of uninduced cells; bands 2-7 show the results on the cells after induction for 3h, 6h, 9h, 12h and 24 hours, respectively. Albumin expression results. It can be seen from Figure 5 that the proSP-B recombinant protein band obtained in the present invention is a single band, no non-specific band appears, and the protein size is consistent with the expectation, which indicates that the expressed protein is proSP-B recombinant protein.

(7) Purification of GST-proSP-B recombinant protein

The recombinant strain pGEX4T-1-proSP-B was inoculated in liquid LB medium containing Amp 50ug/mL at an inoculum size of 1:100, and the cells were collected by centrifugation after induction at 30°C for 12 hours. The bacterial pellet was resuspended in PBS (containing 0.1 mmol/LPMSF), sonicated for 10 min, centrifuged at 12000 r/min for 20 min to collect the supernatant, and then filtered with a 0.45 μm microporous membrane. The protein was purified with a protein purification system, and the purification column was GSTrap FF 16/10 prepacked column. After purification, SDS-PAGE and Western Blot detection and analysis were performed, and the protein concentration was determined with a BCA protein quantification kit. It was determined that the concentration of the recombinant protein was about 1 mg/mL.

The results of SDS-PAGE and Western Blot detection after purification are shown in Figure 6. Specifically, what Fig. 6 (A) shows is the SDS-PAGE result after protein purification, wherein, band 1 is protein Marker; Band 2 shows the bacterium supernatant (purified (before) the result; band 3 shows the result after purification of the bacterial supernatant after induction at 30°C for 12 hours. Figure 6(B) shows the results of Western Blot after protein purification, in which band 1 is the protein marker; band 2 shows the results of uninduced bacterial supernatant; The results after purification of the bacterial supernatant after induction for 12 hours. The results in Fig. 6(A) and Fig. 6(B) show that after purification, a recombinant protein with higher expression (with the amino acid sequence shown in SEQ ID NO: 1) can be obtained.

Example 2

This example is used to illustrate the cytotoxicity of proSP-B recombinant protein provided by the present invention

(1) CCL-149 cells were cultured in R/MINI1640 medium containing 10% FBS.

(2) Culture stable CCL-149 cells, digest the cells with trypsin, collect the cells and dilute them with medium to adjust the cell concentration, so that there are about 5000 cells in 100 μL, inoculate in the 1st to 10th wells of a 96-well plate To 100 μL of CCL-149 cells, 100 μL of medium was added to well 11, and cultured in a 37° C., 5% CO ₂ cell incubator for 24 hours.

(3) Add 10 μL of 1000 μg/mL, 500 μg/mL, 250 μg/mL, 125 μg/mL, 62.5 μg/mL, 21.25 μg/mL, 15.63 μg/mL, 7.81 For μg/mL, 3.91 μg/mL purified protein obtained from step (7) in Example 1, add 10 μL of sterilized distilled water to the 10th and 11th wells.

(4) Place the 96-well plate in a 37° C., 5% CO ₂ cell culture incubator for 12 hours.

(5) Add 10 μL of CCK-8 solution to each well of the 96-well plate.

(6) Incubate the 96-well plate in a 37° C., 5% CO ₂ cell culture incubator for 4 hours.

(7) Measure the absorbance value at 450 nm with a microplate reader.

(8) Calculate the cell viability according to the following formula:

A _C : Absorbance of cells treated with proSP-B recombinant protein solution, that is, the experimental group;

A ₀ : Absorbance without cells, that is, the blank group;

A _S : Absorbance of cells with cells but not treated with proSP-B recombinant protein solution, that is, the control group.

The results of cytotoxicity experiments are shown in Table 1.

Table 1

From the results in Table 1, it can be calculated that the IC ₅₀ of the proSP-B recombinant protein provided by the present invention is 1595.68 μg/mL. Specifically, when the concentration of the proSP-B recombinant protein is 1 mg/mL, the cell viability is 59.62%. It shows that the cytotoxicity of proSP-B recombinant protein is relatively small. According to the cytotoxicity evaluation standard—RGR toxicity grading method, when the relative cell proliferation rate (RGR)≥75%, it can be judged that the drug toxicity is grade 0 or grade 1, when When the RGR is between 50% and 74%, it is judged that the toxicity of the drug is grade 2; grade 2 and below all indicate that the toxicity of the drug is acceptable, that is, it is safe. When the concentration of proSP-B recombinant protein was increased to 1595 μg/mL, the cell proliferation rate was also higher than 50%, indicating that when the concentration of proSP-B recombinant protein was 1595 μg/mL, it was low toxic and could be used for drug development. However, the therapeutic dosage of general drugs is lower than 1595 μg/mL, so the object of this study, proSP-B recombinant protein, is completely safe to be developed as a class of antibacterial drugs.

Example 3

This embodiment is used to illustrate the antibacterial activity of the proSP-B recombinant protein provided by the present invention

1. Activation of strains and preparation of bacterial suspension

(1) Cultivation of Staphylococcus aureus: activate Staphylococcus aureus on LB agar plate by streaking method, culture at 37°C for 12 hours at a constant temperature, after a single colony grows, pick a single colony and inoculate it in 10mL LB liquid medium medium, 37°C, 12h, 150r/min, cultivate, pipette the bacterial solution and sterilized double distilled water to make a bacterial suspension, adjust its concentration, and obtain a bacterial suspension of 10 ⁶ cfu.

(2) Culture of Escherichia coli: activate Escherichia coli on LB agar plate by streaking method, culture at constant temperature for 12 hours at 37°C, after a single colony grows, pick a single colony and inoculate it in 10mL LB liquid medium, 37 ℃, 12h, 150r/min, cultivate, pipette the bacterial solution and sterilized double distilled water to make a bacterial suspension, adjust its concentration, and prepare a bacterial suspension of 10 ⁶ cfu.

(3) Cultivation of Trichophyton rubrum: Prepare SDA medium, pour it into a glass test tube to form an inclined plane, inoculate Trichophyton rubrum at the upper, middle and lower positions of the inclined plane, culture at a constant temperature of 28°C for 10 days, and then pour into each test tube Add 2 mL of sterilized double-distilled water to disperse the Trichophyton rubrum in the tube, adjust its concentration, and prepare a bacterial suspension of 10 ⁶ cfu.

(4) Cultivation of Microsporum gypsumoidus: prepare SDA slant medium plate, inoculate Microsporum gypsumiformis on the upper, middle and lower three positions of the slant, culture at a constant temperature of 28°C for 10 days, and then add sterilized bacteria to each test tube 2mL of double-distilled water to disperse Microsporum gypsum in the tube in the liquid, adjust its concentration, and prepare a bacterial suspension of 10 ⁶ cfu.

(5) Candida albicans: Live Candida albicans on LB agar plate by streaking method, culture at constant temperature for 10 days at 28°C, after a single colony grows, pick a single colony and inoculate it in 10mL LB liquid medium, 37 ℃, 12h, 150r/min, cultivate, pipette the bacterial solution and sterilized double distilled water to make a bacterial suspension, adjust its concentration, and prepare a bacterial suspension of 10 ⁶ cfu.

(6) Pseudomonas aeruginosa: Activate Pseudomonas aeruginosa on LB agar plate by streaking method, culture at 37°C for 12 hours at a constant temperature, after single colony grows, pick a single colony and inoculate it in 10mL LB liquid culture medium, 37°C, 12h, 150r/min, cultivate, pipette the bacterial solution and sterilized double distilled water to make a bacterial suspension, adjust its concentration, and obtain a bacterial suspension of 10 ⁶ cfu.

(7) Methicillin-resistant Staphylococcus aureus: activate the preserved methicillin-resistant Staphylococcus aureus on LB agar plates by streaking method, culture at 37°C for 12 hours at a constant temperature, and pick out after a single colony grows. Inoculate a single colony in 10mL LB liquid medium, culture at 37°C, 12h, 150r/min, pipette the bacterial solution and sterilized double distilled water to make a bacterial suspension, adjust its concentration, and obtain 10 ⁶ cfu bacterial suspension.

2. Antibacterial test of proSP-B recombinant protein-optimization of pH value

(1) Adjust the prepared protein buffer to different pH values to prepare different protein buffers.

(2) Preparation of antibacterial filter paper: Prepare a circular filter paper of about 6mm, sterilize at 121°C for 20 minutes, and dry in an electric blast drying oven for 2 hours. Pick the filter paper pieces with sterile tweezers and soak them in the 500 μg/mL protein solution obtained from step (7) in Example 1, put the soaked filter paper pieces into sterile petri dishes respectively, dry the excess liquid on the surface, To obtain the filter paper soaked in medicine; and use sterile saline as a negative control, and penicillin or terbinafine as a positive control (the positive control of bacteria is penicillin, and the positive control of fungi is terbinafine).

(3) Coating of strains: pipette gun to absorb 50 μL of each diluted and prepared bacterial suspension, and spread evenly on each solid plate with a glass coating rod.

(4) Use sterile tweezers to paste the drug-soaked filter paper, negative control filter paper, and positive control filter paper on the plate, and mark the back of the culture dish with a marker pen.

(5) Cell culture: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus pasted with filter paper were placed upside down in a bacterial biochemical incubator at 37°C for 12 hours; Trichophyton rubrum , Microsporum gypsum, and Candida albicans were placed upside down in a bacterial biochemical incubator at 28°C for 5 days.

(6) Measure the size of the inhibition zone (in mm) of each plate with a ruler, and the results are shown in Table 2. Statistical analysis, screening for proper pH.

Table 2

From the results in Table 2, it can be seen that the proSP-B recombinant protein has certain antibacterial activity at pH 3-8, indicating that the antimicrobial peptide has certain tolerance to weak acidity and weak alkalinity. Among them, in weak acid environment, the antibacterial activity of proSP-B recombinant protein is more obvious, when the pH is 5.6, its antibacterial effect is the strongest, when the pH>5.6, its antibacterial degree increases with the increase of pH value This may be related to the conformational change of the protein, which changes in the molecular conformation in an alkaline environment, which affects the biological activity of the antimicrobial peptide, resulting in a decrease in its antibacterial activity.

3. Determination of antibacterial spectrum of proSP-B recombinant protein

(1) Preparation of antibacterial filter paper: Prepare a circular filter paper of about 6 mm, sterilize at 121°C for 20 minutes, and dry in an electric blast drying oven for 2 hours. Pick the filter paper pieces with sterile tweezers and soak them in the 500 μg/mL protein solution obtained from step (7) in Example 1, put the soaked filter paper pieces into sterile petri dishes respectively, dry the excess liquid on the surface, To obtain the filter paper soaked in medicine; and use sterile saline as a negative control, and penicillin or terbinafine as a positive control (the positive control of bacteria is penicillin, and the positive control of fungi is terbinafine).

(2) Coating of strains: pipette gun to absorb 50 μL of each diluted and prepared bacterial suspension, and spread evenly on each solid plate with a glass coating rod.

(3) Use sterile tweezers to paste the drug-soaked filter paper, negative control filter paper, and positive control filter paper on the plate, and mark the back of the culture dish with a marker pen.

(4) Cell culture: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus pasted with filter paper were placed upside down in a bacterial biochemical incubator at 37°C for 12 hours; Trichophyton rubrum , Microsporum gypsum, and Candida albicans were placed upside down in a bacterial biochemical incubator at 28°C for 5 days.

(5) Measure the size of the inhibition zone on each plate with a ruler, (in mm), the results are shown in Figure 7 (A is a blank control, B is a positive control) and Table 3. Specifically in Fig. 7, Fig. 7 (a) showed the result of Staphylococcus aureus; Fig. 7 (b) showed the result of Escherichia coli; Fig. 7 (c) showed the result of Candida albicans; Fig. 7 (d) shows the results of Trichophyton rubrum; Figure 7(e) shows the results of Microsporum gypsumoid; Figure 7(f) shows the results of Pseudomonas aeruginosa; Figure 7(g) Shown are results for methicillin-resistant Staphylococcus aureus.

Then statistical analysis was carried out to calculate the sensitivity of each bacterium, and the criteria for drug susceptibility testing are shown in Table 4.

table 3

Table 4

Antibacterial zone diameter (mm) over 20 15-10 10-14 Below 10 6 or less sensitivity extremely sensitive high sensitivity Zhongmin Hypoallergenic Not sensitive

It can be seen from Table 3 and Figure 7 that the proSP-B recombinant protein has a strong effect on Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Trichophyton rubrum, Microsporum gypsum-like, and Candida albicans. Inhibitory effect, among which the inhibitory effect on Staphylococcus aureus and methicillin-resistant Staphylococcus aureus is the strongest, and the size of the inhibition zone reaches 20.6mm and 20mm respectively, so Staphylococcus aureus, methicillin-resistant Staphylococcus aureus It is extremely sensitive when the protein concentration is 500μg/mL; while the inhibitory effect on Escherichia coli is strong, and the size of the inhibition zone reaches 15.3mm, so Escherichia coli is highly sensitive to the protein concentration of 500μg/mL; red The antibacterial zone sizes of Trichophyton, Microsporum gypsumoid, and Candida albicans are 12.3mm, 14mm, and 13mm respectively, so when the protein concentration of Trichophyton rubrum, Microsporum gypsumoid, and Candida albicans is 500μg/mL All are highly sensitive; and the inhibition zone size of Pseudomonas aeruginosa is only 8mm, so Pseudomonas aeruginosa is all low-sensitivity when the protein concentration is 500μg/mL.

4. Determination of minimum inhibitory concentration (MIC) of proSP-B recombinant protein

(1) Add 100 μL of the diluted bacterial suspension and culture it in a 96-well plate, and add 50 μL of the protein solution obtained from step (7) in Example 1 to the 10th well in sequence in the 1st to 9th As a blank control, wells 11 and 12 were used as positive controls (penicillin and cefixime for bacteria, terbinafine and ketoconazole for fungi, both at a concentration of 200 μg/mL).

(2) Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus were cultured in a biochemical incubator at 37°C for 12 hours, and then the 96-well cell culture plate was taken out and distributed in the microplate reader. Measure the OD value at a wavelength of 600nm, and calculate the minimum inhibitory concentration of the protein against Staphylococcus aureus, Escherichia coli, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa.

(3) Microsporum gypsumoid, Trichophyton rubrum, and Candida albicans were cultured at a constant temperature of 30°C in a biochemical incubator for 4 days, and the 96-well cell culture plate was taken out, and the OD value was measured at a wavelength of 600nm with a microplate reader, and calculated Minimal inhibitory concentration of protein against Trichophyton rubrum, Microsporum gypsum, and Candida albicans. The results are shown in Table 5.

table 5

Bacteria name Minimum Inhibitory Concentration (MIC) μg/mL Staphylococcus aureus 20 Escherichia coli 30 Candida albicans 60 Trichophyton rubrum 30 Microsporum gypsumoides 30 Pseudomonas aeruginosa 125 MRSA 20

It can be seen from Table 5 that the proSP-B recombinant protein has a high antibacterial effect on bacteria such as Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus, and the concentration of proSP-B recombinant protein that inhibits their growth is higher than that of Low, each only needs 20μg/mL, 30μg/mL, 20μg/mL; and proSP-B recombinant protein also showed obvious antibacterial effect on fungi, such as Candida albicans, Microsporum gypsum, Trichophyton rubrum The minimum inhibitory concentrations are 60μg/mL, 30μg/mL, and 30μg/mL, which are also relatively low, indicating that only a low concentration of proSP-B recombinant protein can inhibit the growth of various bacteria and fungi, thus explaining that proSP-B -B recombinant protein has high bactericidal and antibacterial activity; however, the minimum inhibitory concentration of proSP-B recombinant protein against Pseudomonas aeruginosa is 125 μg/mL, which is relatively higher than that of the other six bacteria. This experiment further proves that proSP-B recombinant protein has a strong inhibitory effect on common G ⁺ , G ^- bacteria and yeast and other fungi.

5. PI determination of the effect of proSP-B recombinant protein on the cell membrane permeability of Staphylococcus aureus

(1) Add 100 μL of the diluted Staphylococcus aureus suspension to columns 1 to 7 of a 96-well plate in sequence for culture, totaling 4 rows. Add 100 μL of sterilized distilled water to column 8, and then add different amounts of proSP-B recombinant protein solution obtained from step (7) in Example 1 to columns 1 to 4 in sequence, so that the final concentration of the protein is 20 μg /mL, 30μg/mL, 60μg/mL, 120μg/mL, add penicillin, Triton X-100, sterilized distilled water, proSP-B recombinant protein solution in sequence from the 5th to the 8th column, so that the final concentration of penicillin is 50μg/mL, The final concentration of Triton X-100 was 0.1%, the final concentration of proSP-B recombinant protein was 120 μg/mL, and finally a certain amount of LB medium was added to ensure that the volume of each well was 200 μL.

(2) Place the 96-well plate in a biochemical incubator at 37° C. for 12 hours, then take it out, and add PI to make the final concentration 50 μg/mL.

(3) After adding PI and incubating for 30 min, measure the fluorescence OD value on a multi-functional microplate reader, the excitation wavelength is 488nm, and the emission wavelength is 630nm.

(4) Calculate and analyze the permeability of bacterial cell membrane with the result of TritonX-100 as 100%.

The results are shown in Table 6.

Table 6

It can be seen from Table 6 that compared with the negative control and the blank control, the fluorescence OD value of the experimental group was significantly higher, and the permeability of the cells was stronger; compared with the positive control, 20 μg/mL of proSP-B recombinant protein had a significant effect on the bacterial cell membrane. The permeability can reach 12.68%, thereby destroying the destruction of the intracellular environment, thereby affecting its growth, so the proSP-B recombinant protein can affect the growth of bacteria by changing the permeability of the cell membrane.

6. Cell membrane extravasation test

(1) Add 100 μL of the prepared Staphylococcus aureus suspension to the 96-well plate in sequence, and add a certain amount of proSP-B recombinant protein obtained in step (7) in Example 1 to 1 to 4 in sequence solution, so that the final concentrations were 20 μg/mL, 30 μg/mL, 60 μg/mL, and 120 μg/mL, and penicillin, Triton X-100, sterilized distilled water, and proSP-B recombinant protein solution were added in sequence from 5 to 8 ° C, where Staphylococcus aureus was not added to the eighth well, so that the final concentration of penicillin was 50 μg/mL, the final concentration of Triton X-100 was 0.1%, the final concentration of proSP-B recombinant protein was 120 μg/mL, and finally a certain amount of LB Culture medium, ensure that the volume of each well is 200 μL.

(2) Place the 96-well plate in a biochemical incubator at 37°C for 12 hours, then take it out, add each reagent in sequence according to the instructions of the LDH kit, and incubate at room temperature for 5 minutes.

(3) Then take it out and measure its OD value at 450nm on a multifunctional microplate reader.

(4) Calculate and analyze the release rate of LDH with the result of TritonX-100 as 100%.

The results are shown in Table 7.

Table 7

It can be seen from Table 7 that under normal cells, the intracellular environment is stable, and the intracellular substances remain relatively stable. However, when the cell membrane is damaged, the cell passage rate increases significantly, and intracellular macromolecular substances will pass through. The damaged cell membrane is released, and the detection of the bacterial culture solution can indirectly indicate whether the permeability of the cell membrane has changed significantly. Lactate dehydrogenase (LDH) is one of the macromolecular substances in the cell. Its detection is relatively simple, and its extravasation can directly reflect the change of the intracellular environment, thereby reflecting the change of the physiological state of the cell. It can be seen from the results of the measurement that compared with the negative control and the blank control, the LDH extravasation of Staphylococcus aureus treated with proSP-B recombinant protein increased significantly, indicating that the cell membrane permeability of the bacteria increased; Compared with the positive control,

120μg/mL proSP-B recombinant protein can make the LDH extravasation rate of cells reach 31%, which in turn affects the stability of bacterial cells and affects their growth, indicating that proSP-B recombinant protein can play a role in changing the physiological state of bacteria.

In general, as can be seen from the results of the above Examples 1-3, the present invention adopts genetic engineering methods, particularly by using specific expression vectors (pGEX4T-1) and expression host bacteria (Escherichia coli BL21 (DE3) pLysS strain), successfully obtained a higher concentration of proSP-B recombinant protein, and, by fusing the GST tag on the pGEX4T-1 vector with proSP-B, since the GST tag is hydrophilic, the proSP-B recombination can be significantly improved The water solubility of protein expands its application range.

In addition, the proSP-B recombinant protein provided by the present invention exhibits good broad-spectrum antibacterial activity, and it has a strong inhibitory effect on common clinical bacteria and fungi, especially Staphylococcus aureus, Escherichia coli, gypsum-like Microsporum, Candida albicans, Trichophyton rubrum, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus showed significant inhibitory activity. Further research found that proSP-B recombinant protein may exert its antibacterial effect by affecting the permeability of bacterial cell membrane, thereby inhibiting the normal growth of bacteria. At the same time, the proSP-B recombinant protein provided by the present invention has less cytotoxicity, even if the protein concentration is increased to 1mg/mL (this concentration is far greater than its MIC value for bacteria), the proliferation ability of the cells is still higher than 59%, This shows that the proSP-B recombinant protein provided by the present invention has higher safety.

The preferred embodiments of the present invention have been described in detail above, however, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the disclosed content of the present invention. All belong to the protection scope of the present invention.

SEQUENCE LISTING

<110> Chongqing University of Technology

<120> Recombinant protein of pulmonary surfactant protein B propeptide and its preparation method and application

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Claims (10)

1. A recombinant protein of pulmonary surfactant protein B propeptide, characterized in that, the recombinant protein has the amino acid sequence shown in SEQ ID NO: 1, or at the amino terminal and/or carboxyl terminal of SEQ ID NO: 1 Link the tagged amino acid sequences. 2. The recombinant protein according to claim 1, wherein the tag is hydrophilic; preferably, the tag is a GST tag. 3. A nucleic acid capable of encoding the recombinant protein of claim 1 or 2. 4. The nucleic acid according to claim 3, wherein the nucleic acid has a nucleic acid sequence shown in SEQ ID NO: 2, or a nucleic acid sequence with a tag attached to the amino terminus and/or carboxyl terminus of SEQ ID NO: 2; Preferably, the tag is hydrophilic; more preferably, the tag is a GST tag. 5. A recombinant plasmid, characterized in that the recombinant plasmid contains the nucleic acid according to claim 3 or 4. 6. The recombinant plasmid according to claim 5, wherein said recombinant plasmid is obtained by inserting the nucleic acid sequence shown in SEQ ID NO:2 between the BamH I and Xho I restriction sites of the pGEX4T-1 plasmid Expression vector. 7. A recombinant bacterial strain, is characterized in that, this recombinant bacterial strain contains the recombinant plasmid described in claim 5 or 6; Preferably, described recombinant bacterial strain is to contain the recombinant plasmid Escherichia coli BL21 (DE3 ) pLysS strain. 8. A method for preparing a recombinant protein of pulmonary surfactant protein B propeptide, characterized in that the method may further comprise the steps: (1) Transforming the recombinant plasmid described in claim 5 or 6 into an expression host bacterium to obtain a recombinant strain; (2) After the recombinant strain is cultured, it is induced and cultivated in the presence of an inducer to obtain a culture medium; (3) Purifying the culture medium obtained in step (2) and removing the label to obtain a recombinant protein of pulmonary surfactant B propeptide. 9. The method according to claim 8, wherein, in step (1), the expression host bacterium is Escherichia coli BL21 (DE3) pLysS strain; Preferably, in step (2), the inducer is isopropyl-β-d-thiogalactoside; Preferably, in step (2), the induction culture conditions include: the temperature is 28-37°C, preferably 28-35°C, more preferably 28-32°C; the time is more than 3h, preferably 3-24h , more preferably 12-24h. 10. the recombinant protein described in claim 1 or 2, the nucleic acid described in claim 3 or 4, the recombinant plasmid described in claim 5 or 6, the recombinant bacterial strain described in claim 7, by claim 8 or 9 described The recombinant protein prepared by the method described above is used in the preparation of antibacterial drugs; preferably, the antibacterial drugs are anti- Staphylococcus aureus , Escherichia coli , Microsporum gypseum , Drugs for at least one of Monilia albican , Trichophyton rubrum , Pseudomonas aeruginosa , and Methicillin -resistant Staphylococcus aureus .