CN117737132A

CN117737132A - Method for enhancing secretion of recombinant receptor protein in insect cells by collagenlike sequence

Info

Publication number: CN117737132A
Application number: CN202311447373.9A
Authority: CN
Inventors: 于博文; 刘志军; 王宁宁
Original assignee: Weifang Medical University
Current assignee: Weifang Medical University
Priority date: 2023-11-02
Filing date: 2023-11-02
Publication date: 2024-03-22

Abstract

The invention belongs to the technical fields of protein engineering and molecular biology, and discloses a method for enhancing secretion of recombinant receptor protein in insect cells by using a collagen-like sequence, synthesizing a screened and optimized SCARA5 collagen-like structural domain sequence and a target protein expression gene, constructing an insect cell expression vector, and obtaining a recombinant vector: optimizing to obtain a nucleotide sequence of a collagen-like sequence coding gene, and determining that the vector is constructed correctly through PCR and sequencing; transferring the vector into DH10Bac escherichia coli competent cells, obtaining escherichia coli successfully recombined through blue and white spot screening, and extracting Bacmid in the escherichia coli; transfecting Bacmid into Sf9 cells, and obtaining P3 generation virus for protein expression; infecting Hi5 cells by using a P3 generation virus, expressing a target protein, and purifying the target protein; the effect of increasing the total amount of secreted protein obtained was monitored. The present invention enhances the secretory expression amount of a secretory receptor protein.

Description

Method for enhancing secretion of recombinant receptor protein in insect cells by collagenlike sequence

Technical Field

The invention belongs to the technical fields of protein engineering and molecular biology, and particularly relates to a method for enhancing secretion of recombinant receptor proteins in insect cells by collagen-like sequences.

Background

At present, the recombinant expression of mammalian cell surface receptor proteins by insect systems is widely applied to biochemical molecular and structural biology research, but the expression and secretion amounts of a plurality of receptor proteins are low. Obtaining enough protein for research or application requires large amount of expression, is high in cost, time-consuming and labor-consuming, and the obtained protein is low in purity, so that the corresponding scientific research and market application of the protein is difficult to support. SCARA5 is a member of the scavenger receptor a family, which contains a collagen-like domain (CL) and a cysteine-rich domain (SRCR) at its carboxy-terminus. It was found by accident during earlier studies of the function of SCARA5 that the SRCR domain of SCARA5 is difficult to secrete when expressed by secretion alone, and the target protein is difficult to obtain, but the secretion amount of the protein secreted by adding the collagen-like domain for co-expression (CL-SRCR) is significantly increased, and the secreted protein is in a monomer form. And the collagen-like domain can spontaneously degrade after secretion, and finally a short peptide is reserved, so that the biochemical property of the SRCR domain is not affected. Based on the findings, the invention relates to the optimized and modified collagen-like peptide, and the collagen-like peptide is applied to express and secrete various target receptor proteins in an insect cell system.

Through the above analysis, the problems and defects existing in the prior art are as follows: when a part of the secretory receptor protein is secreted and expressed by an insect expression system, the expression quantity is low, and the requirements of related researches are difficult to meet.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention provides a method for enhancing secretion of recombinant receptor proteins in insect cells by collagen-like sequences.

The invention is realized by a method for enhancing the secretion of recombinant receptor protein in insect cells by a collagen-like sequence, wherein the method for enhancing the secretion of the recombinant receptor protein in the insect cells by the collagen-like sequence comprises the following steps:

firstly, synthesizing an optimized SCARA5 collagen domain sequence and a target protein expression gene which are subjected to mutation screening, and constructing the optimized SCARA5 collagen domain sequence and the target protein expression gene into an insect cell expression vector according to the sequence of a signal peptide, the collagen domain sequence and the target gene to obtain a recombinant vector;

secondly, transferring the vector into DH10Bac escherichia coli competent cells, obtaining escherichia coli successfully recombined through blue and white spot screening, and extracting Bacmid in the escherichia coli;

thirdly, transfecting the Bacmid into Sf9 cells, packaging baculovirus, and obtaining P3 generation virus for protein expression;

step four, infecting Hi5 cells by the P3 generation virus, expressing the target protein, and purifying the target protein by an affinity purification gel chromatographic method;

fifth, the effect of increasing the total amount of secreted protein obtained was monitored.

Further, the amino acid sequence of the SCARA5 optimized in the first step is shown as SEQ ID NO:1, the specific mutated residues of the sequence after screening and optimization compared with the original type gene are K7L, K12A, R34A and D79L respectively. The nucleotide sequence of the collagen-like sequence encoding gene is obtained through adding a stop codon and codon optimization, and the correct construction of the vector is determined through PCR and sequencing.

Further, in the first step, the optimized collagenlike domain and the encoding gene of the target protein are synthesized through genes, then the optimized collagenlike domain and the encoding gene of the target protein are connected to a gene fragment by using high-fidelity enzyme through an overlap extension PCR method, wherein the collagenlike domain is positioned at the amino end of the target gene, the target gene fragment obtained by PCR is recovered through agarose gel, and the recovered gene fragment is connected with a pFastBac-melitten insect cell expression vector containing a signal peptide, for example, so that the expression sequence of the protein is the signal peptide-collagenlike domain-target protein, and finally the correctness of the inserted fragment is verified through sequencing.

Further, in the second step, 20ng of the recombinant plasmid is added into DH10Bac competent cells, after uniform mixing, the mixture is kept stand on ice for 15min and then subjected to heat shock at 42 ℃ for 60s, and then 1ml of antibiotic-free LB culture is added for resuscitation at 220rpm based on shaking at 37 ℃ for 4hr; finally, the mixture was divided into 20. Mu.l, 10. Mu.l and 5. Mu.l, and the mixture was applied to a three-antibody LB plate (containing 100. Mu.g/ml X-gal, 50. Mu.g/ml Kan) ⁺ ，10μg/ml Tet ⁺ ，7μg/ml Gen ⁺ 1mM IPTG), the plates were incubated in an incubator at 37℃for about two days. Picking up monoclonal white fungusFalls into 3mL of three-antibody LB medium (50. Mu.g/mL Kan) ⁺ ，10μg/ml Tet ⁺ ，7μg/ml Gen ⁺ ) Shake culturing at 37deg.C for 10hr, and extracting Bacmid.

Further, bacmid was extracted by: 12000g of the bacterial liquid was collected, 150. Mu.l of solution I (25 mM glucose, 150mM NaCl,25mM Tris,pH 8.0) was added to resuspend the bacterial cells, 200. Mu.l of solution II (0.2M NaOH,1% SDS) was added to gently mix them, and after standing for about 5min until the bacterial liquid was completely lysed, 200. Mu.l of solution III (1.5M HAC,1M KAC) was added to gently mix them. 14000g was centrifuged for 15min, 450. Mu.l of supernatant was transferred to a fresh tube, and 2 volumes of absolute ethanol were added and mixed well and allowed to stand at-20℃for 2hr. Subsequently, 14000g was centrifuged for 15min, the supernatant was discarded, and the ethanol was dried in a metal bath at 55 ℃. The Bacmid was dissolved by standing for 15min with 80. Mu.l double distilled water, and then the nucleic acid concentration was measured by a spectrophotometer. 0.5 μl of Bacmid is taken, whether the fragment is inserted or not is identified by PCR, the correct Bacmid is filtered and sterilized, and the fragment is stored at 4 ℃ for standby.

Further, the third step of baculovirus packaging process is to take Sf9 cells growing in log phase, spread the Sf9 cells in a 6-well plate, and the density is about 0.8X10 ⁶ And/or holes. After the cells were attached to the bottom of the 6-well plate (about 15 min), the supernatant medium was aspirated, 2mL Grace medium (Invitrogen) was added to each well, and placed in a 27℃cell incubator. Mu.l Grace medium was added to a sterile EP tube, 7. Mu.l Cellfection II was added and mixed by vortexing and standing for about 30min. Mu.g of Bacmid was dissolved in 100. Mu.l of Grace medium and mixed with Cellfection II solution and left for 25min. The mixture was added to a 6-well plate, left for about 4hr, and the medium was washed off, and SF900 II insert medium was added. After about 5 days, the supernatant was collected. And taking a small amount of supernatant to carry out Western blotting identification, amplifying and preserving the P1 generation virus successfully packaged.

Further, preparing the P2 generation virus, taking T25 flash, adding 4ml of ESF921 complete medium into each bottle, adding Sf9 cells, shaking slightly by hand to enable the cells to be dispersed uniformly, placing the cells in an incubator until the bottom of the cells are paved with 80%, removing redundant suspended cells, adding 4ml of ESF921 complete medium, adding 500 mu l of P1 generation virus, placing the cells in an insect incubator, placing the P2 generation virus for about 4 days, carrying out Western detection after cell lysis, sampling and centrifuging, and confirming whether the target protein is generated. Collecting P2 generation virus, taking T75 flash, adding 8ml of ESF921 complete culture medium into each bottle, spreading cells, adding 500 μl of P2 generation virus, mixing, culturing for 4 days, observing cell lysis state, collecting P3 generation virus, and temporarily storing at 4deg.C. The collected P3 generation virus is sampled to carry out Western-Blot experiment, and the increase of the protein expression amount added into the collagen-like structural domain is compared.

Further, the High-Five cells are continuously expanded in the fourth step, and cultured to 1L,300-400 ml/bottle, and the dilution density is 2.5x10 ⁶ About 2ml of the P3 generation virus was added to each cell/ml, and the mixture was shake-cultured for 3 days. After the protein expression was completed, it was removed from the cell house shaker, poured into a clean beaker, split-filled into centrifuge tubes and trimmed, centrifuged at 13000g for 20min, and the supernatant was collected, taking care not to pour out the cell pellet. The sample was taken before purification to carry out Western-Blot experiments to compare the increase in protein expression levels with the addition of the collagen-like domains.

Further, the expression of the fluorescent protein is directly analyzed by comparing the fluorescent intensity values by a detection enzyme-labeled instrument.

Further, purifying the target protein by an affinity purification mode, purifying the obtained supernatant by a peristaltic pump through Ni Smart Beads at a flow rate of 0.5ml/min, and eluting the target protein by a TBS solution containing 5mM imidazole and 250mM imidazole; concentrating the obtained protein by using an ultrafiltration tube, desalting, concentrating to 1ml, taking 20 mu l of sample, detecting by SDS-PAGE electrophoresis and coomassie brilliant blue staining, judging the protein purity, and primarily judging the total amount of the comparative protein; the resulting protein was then purified by AKTA gel chromatography, specifically using GE HiLoad Superdex PG for separation, and the corresponding protein peak was collected for concentration, and the amount of the resulting protein was calculated by the absorbance of a280 combined with the extinction coefficient and compared to the experimental group without the addition of the collagen-like domain.

In combination with the technical scheme and the technical problems to be solved, the technical scheme to be protected has the following advantages and positive effects:

first, in the study or application of extracellular domain of mammalian cell surface receptor, recombinant proteins are often required, but many proteins suffer from low expression levels even by secretion of higher levels of expression by insect cell baculovirus expression systems, e.g., the SRCR domain of SCARA5 is secreted directly at a yield of less than 3mg/L. According to the invention, on the basis that the SCARA5 collagen domain is added after the signal peptide discovered in the early stage to promote the secretion expression of the receptor protein, the optimized SCARA5 collagen domain is obtained through alanine and leucine saturation mutation screening, and the secretion capacity of the target protein can be further enhanced to 2-7 times of that of the control group protein by the optimized SCARA5 collagen domain. And collagen-like substances can be spontaneously degraded after secretion, so that the influence on the biochemical experiment and the structural biological research of target proteins is small. In sum, by fusing the optimized SCARA5 collagen domain after secretion of the signal peptide, the secretion amount of a series of receptor proteins can be remarkably increased, and the efficiency of related protein research and the yield of functional proteins can be improved.

Secondly, the invention speculates that the collagen-like structural domain promotes the expression and secretion of target protein, and further screens out a modified SCARA5 collagen-like structural domain through saturation mutation of alanine and leucine, and tests the efficacy of the modified SCARA5 collagen-like structural domain in the expression of different receptor structural domains. According to the invention, the secretory expression quantity of the secretory receptor protein is enhanced by adding a section of optimized collagen-like structural domain amino acid sequence after the signal peptide commonly used in the insect expression system, and the method for adding the optimized collagen-like structural domain is simple and convenient, and has high application value.

Thirdly, the expected benefits and commercial value after the technical scheme of the invention is converted are as follows: the invention is applied to the production of commercial recombinant protein, can obviously reduce the production cost of the protein by 2-7 times, and enhances the competitiveness of related products.

The technical scheme of the invention fills the technical blank in the domestic and foreign industries: means for enhancing secretion of secreted proteins have been reported to include engineering signal peptides with the addition of a secretagogue nucleic acid sequence in the forepart of the signal peptide. The technology of increasing collagen-like structural domain after secretion of protein signal peptide and enhancing secretion expression of target protein has not been reported at home and abroad. The method for increasing the collagen-like domain has higher secretion expression amount of the protein than the prior method.

Fourth, the following is a significant technological advance brought by the method of enhancing secretion of recombinant receptor proteins in insect cells by such collagen sequences:

1) Optimizing protein expression: by using the optimized SCARA5 collagen domain sequence, the expression level of the target protein in insect cells is improved. This has a crucial role for large-scale production and use of the protein of interest.

2) And (3) improving protein stability: collagen-like domains generally help to improve protein stability in proteins. Therefore, the collagen-like sequence is fused into the target protein, so that the stability of the protein can be improved, and the protein is more tolerant in the subsequent purification and application processes.

3) Enhancing protein secretion: the insect cells are used as expression hosts, and the characteristics of high density, high expression quantity and correct protein secretion and folding of the insect expression system are utilized, so that the secretion, collection and purification efficiency of the protein is improved.

4) The production efficiency is improved: through specially designed and optimized expression vectors and expression steps, the efficiency of producing target proteins by insect cells can be greatly improved.

5) Increased flexibility: this method offers the possibility of expressing and secreting various recombinant proteins in insect cells with a high flexibility of application.

Therefore, the method has important application value in the fields of protein engineering and biopharmaceuticals, and can promote the technical progress of the related fields.

Drawings

FIG. 1 is a flow chart of a method for enhancing secretion of recombinant receptor proteins in insect cells using collagen-like sequences provided in an embodiment of the present invention;

FIG. 2 is a schematic diagram of a method for enhancing secretion of recombinant receptor proteins in insect cells using collagen-like sequences according to an embodiment of the present invention;

FIG. 3 is a schematic diagram of a Western-Bolt experiment performed by taking a P3 generation virus according to the embodiment of the invention;

FIG. 4 is a schematic diagram showing the comparison of the addition of a primary collagen-like peptide and the optimization of the expression of an altered collagen-like peptide according to an embodiment of the present invention;

FIG. 5 is a SDS-PAGE Coomassie brilliant blue staining pattern of the collagen-like domain after 48hr at 4deg.C provided by the present examples;

FIG. 6 is a graph showing comparison of protein production by secretory expression with respect to a collagen-like domain not added according to an embodiment of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

As shown in fig. 1, the method for enhancing secretion of recombinant receptor protein in insect cells by using collagen-like sequences provided by the embodiment of the invention comprises the following steps:

s101: synthesizing a screened and optimized SCARA5 collagen domain sequence and a target protein expression gene, constructing an insect cell expression vector, and obtaining a recombinant vector: the amino acid sequence of the modified and optimized SCARA5 is shown as SEQ ID NO:1, adding a stop codon and optimizing the codon to obtain a nucleotide sequence of a collagen-like sequence coding gene, and determining that the vector is constructed correctly through PCR and sequencing;

s102: transferring the recombinant plasmid into DH10Bac competent cells, screening by blue white to obtain strains which are successfully recombined into a Bacmid genome, and extracting Bacmid from the strains;

s103: transfecting the Bacmid into Sf9 cells, packaging baculovirus, and obtaining P3 generation virus for protein expression;

s104: infecting Hi5 cells by using P3 generation virus, expressing target protein, and purifying the target protein by means of affinity purification gel chromatography and the like;

s105: the effect of increasing the total amount of secreted protein obtained was monitored.

In the embodiment of the present invention, step S101, the optimized collagenlike domain and the encoding gene of the target protein are synthesized by gene synthesis, then the optimized collagenlike domain and the encoding gene of the target protein are connected to a gene fragment by using high-fidelity enzyme through an overlap extension PCR method, wherein the collagenlike domain is located at the amino end of the target gene (fig. 2), the target gene fragment obtained by PCR is recovered by agarose gel, and the recovered gene fragment is connected with, for example, pFastBac-melanten insect cell expression vector containing signal peptide, so that the expression sequence of the protein is the signal peptide-collagenlike domain-target protein, and finally the correctness of the insert fragment is verified by sequencing (fig. 2).

In the embodiment of the invention, step S102, adding 20ng of recombinant plasmid into DH10Bac competent cells, uniformly mixing, standing on ice for 15min, then carrying out heat shock at 42 ℃ for 60S, and then adding 1ml of antibiotic-free LB culture for resuscitation at 220rpm based on shaking at 37 ℃ for 4hr; finally, the mixture was divided into 20. Mu.l, 10. Mu.l and 5. Mu.l, and the mixture was applied to a three-antibody LB plate (containing 100. Mu.g/ml X-gal, 50. Mu.g/ml Kan) ⁺ ，10μg/ml Tet ⁺ 7. Mu.g/ml Gen+,1mM IPTG) and plates were incubated in an incubator at 37℃for about two days. A monoclonal white colony was picked up in 3mL of triple antibody LB medium (50. Mu.g/mL Kan) ⁺ ，10μg/ml Tet ⁺ ，7μg/ml Gen ⁺ ) Shake culturing at 37deg.C for 10hr, and extracting Bacmid. Bacmid was extracted by: 12000g of the bacterial liquid was collected, 150. Mu.l of solution I (25 mM glucose, 150mM NaCl,25mM Tris,pH 8.0) was added to resuspend the bacterial cells, 200. Mu.l of solution II (0.2M NaOH,1% SDS) was added to gently mix them, and after standing for about 5min until the bacterial liquid was completely lysed, 200. Mu.l of solution III (1.5M HAC,1M KAC) was added to gently mix them. 14000g was centrifuged for 15min, 450. Mu.l of supernatant was transferred to a fresh tube, and 2 volumes of absolute ethanol were added and mixed well and allowed to stand at-20℃for 2hr. Subsequently, 14000g was centrifuged for 15min, the supernatant was discarded, and the ethanol was dried in a metal bath at 55 ℃. The Bacmid was dissolved by standing for 15min with 80. Mu.l double distilled water, and then the nucleic acid concentration was measured by a spectrophotometer. 0.5 μl of Bacmid is taken, whether the fragment is inserted or not is identified by PCR, the correct Bacmid is filtered and sterilized, and the fragment is stored at 4 ℃ for standby.

In the embodiment of the present invention, step S103, a packaging process of baculovirus is performed, and Sf9 cells growing in log phase are takenSpread in 6-well plate with density of about 0.8X10 ⁶ And/or holes. After the cells were attached to the bottom of the 6-well plate (about 15 min), the supernatant medium was aspirated, 2mL Grace medium (Invitrogen) was added to each well, and placed in a 27℃cell incubator. Mu.l Grace medium was added to a sterile EP tube, 7. Mu.l Cellfection II was added and mixed by vortexing and standing for about 30min. Mu.g of Bacmid was dissolved in 100. Mu.l Grace's insect cell culture medium (Invitrogen) and mixed with Cellfection II solution and left for 25min. The mixture was added to a 6-well plate, left for about 4hr, and the medium was washed off, and SF900 II insert medium was added. After about 5 days, the supernatant was collected. And taking a small amount of supernatant to carry out Western blotting identification, amplifying and preserving the P1 generation virus successfully packaged.

Preparing P2 generation virus, taking T25 flash, adding 4ml of ESF921 complete culture medium into each bottle, adding Sf9 cells, shaking slightly by hand to enable the cells to be dispersed uniformly, placing the cells into an incubator until the cells are full of about 80% of the bottom, removing redundant suspended cells, adding 4ml of ESF921 complete culture medium, adding 500 μl of P1 generation virus, placing the cells into an insect incubator, performing Western detection after sampling and centrifugation, and confirming whether target proteins are generated. Collecting P2 generation virus, taking T75 flash, adding 8ml of ESF921 complete culture medium into each bottle, spreading cells, adding 500 μl of P2 generation virus, mixing, culturing for 4 days, observing cell lysis state, collecting P3 generation virus, and temporarily storing at 4deg.C. The collected P3 generation virus is sampled to carry out Western-Blot experiment, and the increase of the protein expression amount added into the collagen-like structural domain is compared.

In the embodiment of the invention, step S104, the High-Five cells are continuously amplified and cultured to 1L,300-400 ml/bottle, and the dilution density is 2.5x10 ⁶ About 2ml of the P3 generation virus was added to each cell/ml, and the mixture was shake-cultured for 3 days. After the protein expression was completed, it was removed from the cell house shaker, poured into a clean beaker, split-filled into centrifuge tubes and trimmed, centrifuged at 13000g for 20min, and the supernatant was collected, taking care not to pour out the cell pellet. The sample was taken before purification to carry out Western-Blot experiments to compare the increase in protein expression levels with the addition of the collagen-like domains. The expression of the fluorescent protein can be directly analyzed by comparing the fluorescent intensity values by a detection enzyme-labeled instrument.

The target protein was purified by affinity purification, the supernatant obtained above was purified by a peristaltic pump through Ni Smart Beads (Dimmalia and Biotechnology) at a flow rate of 0.5ml/min, and then eluted by a TBS solution containing 5mM imidazole, 250mM imidazole. Then concentrating the obtained protein by using an ultrafiltration tube, desalting, concentrating to 1ml, taking 20 mu l of sample, detecting by SDS-PAGE electrophoresis and coomassie brilliant blue staining, judging the protein purity, and primarily judging the total amount of the comparative protein. The resulting protein was then purified by AKTA (GE healthcare) gel chromatography, separated using GE HiLoad Superdex200PG, the corresponding protein peak was collected for concentration, and the amount of protein finally obtained was calculated by the absorbance of a280 combined with the extinction coefficient and compared to the experimental group for addition of the collagen-like domain.

The specific yield comparisons of the CTLD domain of human SCARA4, SCARA5, SRCR domain of MARCO and the two SRCR2-3 domains of CD163 purified in the above manner with and without the addition of collagenous domains are shown in FIG. 6.

Example 1

As shown in FIG. 2, the method for enhancing secretion of recombinant receptor protein in insect cells by using collagen-like sequences provided by the embodiment of the invention is a specific flow for expressing SRCR domain of human MARCO protein, and comprises the following steps:

step one, synthesizing a coding gene (synthesized from a worker) of a SRCR domain of a human MARCO protein, as shown in SEQ ID NO:3, performing overlap extension PCR on the coding gene, a signal peptide and an optimized collagen-like domain to obtain a DNA chain, then performing glue recovery, and constructing a vector containing the signal peptide between BamHI/Xball of a pFastBac-melitten vector by homologous recombination, wherein the sequence of the pFastBac-melitten vector is shown in SEQ ID NO: 4;

specifically, the coding gene of SRCR domain of MARCO protein is amplified by using high-fidelity enzyme, and the amplification primer pair is MARCO F (shown as SEQ ID NO: 5) and MARCO R (shown as SEQ ID NO: 6). Meanwhile, the collagen-like domain gene is amplified by using Signal F (shown as SEQ ID NO: 7) and CL R (shown as SEQ ID NO: 8), and after the two amplified products are recovered by glue, the two amplified products are used as templates to carry out secondary amplification by using the two primers of Signal F and MARCO R. Simultaneously, the pFastBac-melitten vector was subjected to double digestion by BamHI and XbaII, the two products were subjected to gel recovery, and then both were constructed into recombinant vectors by homologous recombination. In addition to this, expression constructs not containing collagenlike domains were constructed in a similar manner as control.

And secondly, transferring the vector into DH10Bac escherichia coli competent cells, screening to obtain escherichia coli successfully recombined through blue and white spots, and extracting Bacmid from the escherichia coli, wherein the specific flow is the same as that described in the summary of the invention.

And thirdly, transfecting the Bacmid into Sf9 cells, packaging baculovirus, and obtaining the virus of the generation P3 for protein expression, wherein the specific flow is the same as that described in the summary of the invention. The obtained P3-generation virus was subjected to Western-Blot experiments in which 15. Mu.l of each of the control group and the collagenous group was used to detect the target protein amount by His tag, and the result showed that the collagen-like group had a protein amount about 4 times that of the control group (FIG. 3).

Step four, infecting Hi5 cells by the P3 generation virus, expressing target protein, purifying the target protein by means of affinity purification gel chromatography and the like, wherein the specific flow is the same as that of the process described in the invention content, 1000ml of cells in the same time are expressed in each of an experimental group and a control group, SDS-PAGE and Coomassie brilliant blue staining experiments are carried out on samples with the same volume after purification, and the increase of the protein expression amount added into the collagen-like domain is compared, so that the result shows that the protein amount of the collagen-like group is about 4 times that of the control group (figure 4).

Step five, before gel chromatography purification, the SRCR protein is left at 4 ℃ for 48hr to fully degrade the collagen-like domain (figure 5), then gel chromatography purification is carried out to obtain high-purity MARCO SRCR protein after gel chromatography purification, concentration is carried out, and the total amount of finally obtained protein is judged through A280 absorption and extinction coefficient, so that the collagen-like experimental group obtains about 15.5mg SRCR protein, and the control group obtains about 3.3mg SRCR protein (figure 6).

SEQ ID NO:1

KGPPGPLGDQGAEGKEGRPGIPGLPGLRGLPGEAGTPGLPGPKGDDGKLGATGPMGMRGFKGDRGPKGEKGEKGDRAGL

SEQ ID NO:2：

ATGAAATTCT TAGTCAACGT TGCCCTTGTT TTTATGGTCGTATACATTTC TTACATCTAT

GCGGCGGCGA AAGGACCGCC CGGCCCATTG GGAGATCAGGGAGCAGAAGG CAAAGAGGGT

CGCCCAGGCA TTCCCGGCTT GCCAGGTTTG AGGGGCCTGCCTGGCGAAGC TGGAACACCG

GGCCTGCCGG GACCAAAGGG CGATGATGGC AAGCTGGGAGCCACGGGCCC TATGGGTATG

CGGGGCTTCA AGGGCGACCG GGGACCGAAG GGAGAAAAGGGCGAGAAAGG CGATCGTGCG

GGACTG

SEQ ID NO:3：

GTTCGCATCG TAGGAAGTTC GAATCGTGGC CGCGCAGAGGTGTATTATAG CGGTACCTGG GGCACGATCT

GCGATGATGA GTGGCAGAAC AGCGATGCGA TCGTTTTCTGCCGCATGCTG GGTTACAGTA AAGGACGAGC

CCTGTATAAG GTTGGCGCAG GAACCGGACA GATCTGGCTGGACAACGTGC AATGCCGCGG TACAGAATCG

ACCCTCTGGT CCTGCACAAA GAACTCCTGG GGCCATCACGATTGCAGCCA TGAGGAAGAC GCAGGAGTCG

AGTGCTCC

SEQ ID NO:4：

GACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCC

TGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGT

GGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCT

TTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAG

CTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTAC

AATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTT

ATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTG

ATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATT

TCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGC

TCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGT

GCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGA

GAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCT

GCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCG

GTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTC

ACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTG

CTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACG

ATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCA

TGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAA

ACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCG

CAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAA

TAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC

CTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGG

GTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTA

TCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT

AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTC

AGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTA

ATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAA

TCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAG

ATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTG

CAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGA

GCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATAC

CAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAAC

TCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCT

GCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATA

GTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACA

CAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGC

GTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAG

GTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTT

CCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCT

CTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTAT

GGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG

CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACC

GTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACC

GAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGT

ATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAGACCAGCCGCGT

AACCTGGCAAAATCGGTTACGGTTGAGTAATAAATGGATGCCCTGCGTAA

GCGGGTGTGGGCGGACAATAAAGTCTTAAACTGAACAAAATAGATCTAA

ACTATGACAATAAAGTCTTAAACTAGACAGAATAGTTGTAAACTGAAATC

AGTCCAGTTATGCTGTGAAAAAGCATACTGGACTTTTGTTATGGCTAAAG

CAAACTCTTCATTTTCTGAAGTGCAAATTGCCCGTCGTATTAAAGAGGGG

CGTGGCCAAGGGCATGGTAAAGACTATATTCGCGGCGTTGTGACAATTTA

CCGAACAACTCCGCGGCCGGGAAGCCGATCTCGGCTTGAACGAATTGTTA

GGTGGCGGTACTTGGGTCGATATCAAAGTGCATCACTTCTTCCCGTATGC

CCAACTTTGTATAGAGAGCCACTGCGGGATCGTCACCGTAATCTGCTTGC

ACGTAGATCACATAAGCACCAAGCGCGTTGGCCTCATGCTTGAGGAGATT

GATGAGCGCGGTGGCAATGCCCTGCCTCCGGTGCTCGCCGGAGACTGCGA

GATCATAGATATAGATCTCACTACGCGGCTGCTCAAACCTGGGCAGAACG

TAAGCCGCGAGAGCGCCAACAACCGCTTCTTGGTCGAAGGCAGCAAGCG

CGATGAATGTCTTACTACGGAGCAAGTTCCCGAGGTAATCGGAGTCCGGC

TGATGTTGGGAGTAGGTGGCTACGTCTCCGAACTCACGACCGAAAAGATC

AAGAGCAGCCCGCATGGATTTGACTTGGTCAGGGCCGAGCCTACATGTGC

GAATGATGCCCATACTTGAGCCACCTAACTTTGTTTTAGGGCGACTGCCCT

GCTGCGTAACATCGTTGCTGCTGCGTAACATCGTTGCTGCTCCATAACATC

AAACATCGACCCACGGCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCAT

AGACTGTACAAAAAAACAGTCATAACAAGCCATGAAAACCGCCACTGCG

CCGTTACCACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGAGCG

CATACGCTACTTGCATTACAGTTTACGAACCGAACAGGCTTATGTCAACT

GGGTTCGTGCCTTCATCCGTTTCCACGGTGTGCGTCACCCGGCAACCTTGG

GCAGCAGCGAAGTCGAGGCATTTCTGTCCTGGCTGGCGAACGAGCGCAA

GGTTTCGGTCTCCACGCATCGTCAGGCATTGGCGGCCTTGCTGTTCTTCTA

CGGCAAGGTGCTGTGCACGGATCTGCCCTGGCTTCAGGAGATCGGAAGAC

CTCGGCCGTCGCGGCGCTTGCCGGTGGTGCTGACCCCGGATGAAGTGGTT

CGCATCCTCGGTTTTCTGGAAGGCGAGCATCGTTTGTTCGCCCAGGACTCT

AGCTATAGTTCTAGTGGTTGGCTACGTATACTCCGGAATATTAATAGATC

ATGGAGATAATTAAAATGATAACCATCTCGCAAATAAATAAGTATTTTAC

TGTTTTCGTAACAGTTTTGTAATAAAAAAACCTATAAATATTCCGGATTAT

TCATACCGTCCCACCATCGGGCGCGGATCCCGGTCCGAAGCGCGCGGAAT

TCAAAGGCCTACGTCGACGAGCTCACTAGTCGCGGCCGCTTTCGAATCTA

GACATCACCATCACCATCACTAGGCCTGCAGTCTCGAGGCATGCGGTACC

AAGCTTGTCGAGAAGTACTAGAGGATCATAATCAGCCATACCACATTTGT

AGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGA

AACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATA

ATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTT

TTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATC

ATGTCTGGATCTGATCACTGCTTGAGCCTAGGAGATCCGAACCAGATAAG

TGAAATCTAGTTCCAAACTATTTTGTCATTTTTAATTTTCGTATTAGCTTAC

GACGCTACACCCAGTTCCCATCTATTTTGTCACTCTTCCCTAAATAATCCT

TAAAAACTCCATTTCCACCCCTCCCAGTTCCCAACTATTTTGTCCGCCCAC

AGCGGGGCATTTTTCTTCCTGTTATGTTTTTAATCAAACATCCTGCCAACT

CCATGTGACAAACCGTCATCTTCGGCTACTTTTTCTCTGTCACAGAATGAA

AATTTTTCTGTCATCTCTTCGTTATTAATGTTTGTAATTGACTGAATATCAA

CGCTTATTTGCAGCCTGAATGGCGAATGG

SEQ ID NO:5：

GCGATCGTGCGGGACTGGTTCGCATCGTAGGAAGTTCGA

SEQ ID NO:6：

TGGTGATGGTGATGTCTAGAGGAGCACTCGACTCCTGC

SEQ ID NO:7：

GTCCCACCATCGGGCGCATGAAATTCTTAGTCAACGTTGC

SEQ ID NO:8：

CAGTCCCGCACGATCGC

The specific procedure of example 1 was developed by the method of the present invention, except that the CTLD domain of human-derived SCARA4, the SRCR domain of SCARA5 and the two SRCR2-3 domains of CD163 were purified in a similar manner, and the yields of the added optimized collagen-like domain and the non-added collagen-like domain were compared, as shown in fig. 6, and the addition of the optimized collagen-like domain generally improved the yield of the target protein by about 2-7 times.

In the examples, the effect of the optimized collagen-like peptide on promoting MARCO SRCR secretion was first verified, and the results show that the optimized collagen-like peptide can promote SRCR secretion more than the original collagen-like peptide (FIG. 3 and FIG. 4), and in addition, the optimized collagen-like peptide is similar to the original collagen-like peptide, and can be gradually degraded, so that the influence on the target protein is reduced (FIG. 5). The CTLD domain of SCARA4, the SRCR domain of SCARA5 and the two continuous domains SRCR2-3 of CD163 were expressed by a similar method, and the yields of the added optimized collagen-like domain and the non-added collagen-like domain were compared, as shown in FIG. 6, and the addition of the optimized collagen-like domain generally increased the yield of the target protein by about 2-7 times.

The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.

Claims

1. A method for enhancing secretion of a recombinant receptor protein in an insect cell by a collagen-like sequence comprising the steps of:

firstly, synthesizing a screened and optimized SCARA5 collagen domain sequence and a target protein expression gene, and constructing an insect cell expression vector to obtain a recombinant vector;

2. The method for enhancing secretion of recombinant receptor protein in insect cells by collagen-like sequence according to claim 1, wherein the amino acid sequence of SCARA5 optimized in the first step is as set forth in SEQ ID NO:1, adding a stop codon and optimizing the codon to obtain a nucleotide sequence of a collagen-like sequence coding gene, and determining that the vector is constructed correctly through PCR and sequencing; the residues of the specific mutations compared with the original type gene are K7L, K12A, R34A and D79L, respectively.

3. The method for enhancing secretion of recombinant receptor protein in insect cells by using collagen-like sequence according to claim 1, wherein the optimized collagen-like domain and the encoding gene of target protein are synthesized by gene, then the optimized collagen-like domain and the encoding gene of target protein are connected to a gene fragment by using high-fidelity enzyme through overlapping extension PCR method, wherein the collagen-like domain is positioned at the amino end of the target gene, the target gene fragment obtained by PCR is recovered by agarose gel, the recovered gene fragment is connected with insect cell expression vector containing signal peptide, so that the expression sequence of the protein is signal peptide-collagen-like domain-target protein, and finally the correctness of the inserted fragment is verified by sequencing; the expression vector is a pFastbac series vector containing a signal peptide, and the signal peptide is not limited to melitten.

4. The method of claim 1, wherein the packaging of the virus is performed by a baculovirus expression system using host cells comprising Sf9, hi5 cells.

5. The method for enhancing secretion of recombinant receptor protein in insect cells by using collagen-like sequence according to claim 1, wherein after packaging of the P3-generation virus is completed, the sample of the virus is sampled for Western-Blot experiment, and the increase of the protein expression amount added with collagen-like domain is compared.

6. The method for enhancing secretion of recombinant receptor protein in insect cells by using collagen-like sequence according to claim 1, wherein the protein expression is completed, the supernatant is collected and sampled before purification for Western-Blot experiment, and the increase of the protein expression amount added to the collagen-like domain is compared.

7. The method for enhancing secretion of recombinant receptor protein in insect cells by using collagen-like sequence according to claim 1, wherein after the protein expression is completed, the target protein is purified by an affinity purification mode comprising His, GST, MBP and other labels, and the purified sample is detected by SDS-PAGE electrophoresis and Coomassie brilliant blue staining to judge the protein purity, and the total amount of the comparative protein is preliminarily judged; the resulting protein was then purified by AKTA gel chromatography, the corresponding protein peak was collected for concentration, and the amount of the resulting protein was calculated by combining the absorbance of a280 with the extinction coefficient and compared to the experimental group for addition of the collagen-like domain.

8. The method for enhancing secretion of recombinant receptor protein in insect cells by using collagen-like sequence according to claim 1, wherein the expression of fluorescent protein is directly analyzed by comparing the fluorescence intensity values by a detection microplate reader.