CN108486123B - Modified human lactoferrin gene suitable for expression in silk gland of Bombyx mori and its expression system and application - Google Patents

Abstract

The invention relates to a modified human lactoferrin gene suitable for expression in silk glands of Bombyx mori and its expression system and application. The modified human lactoferrin gene is shown in SEQ ID NO.1, and the encoded amino acid is shown in SEQ ID NO.2 As shown, the modified human lactoferrin gene was combined with the promoter of secreted sericin 1 gene and the terminator of secreted sericin 1 gene to form an expression frame, and the enhancer hr3 was used to enhance the expression, and the piggyBac transposable arm and fluorescent selection marker were connected at the same time. The gene constructs an expression system, the expression system can efficiently express recombinant human lactoferrin in silk glands of silkworm, has biological activity, can be used for large-scale production of recombinant human lactoferrin, and has a good market prospect.

Description

Modified human lactoferrin gene suitable for expression in silk gland of silkworm and its expression system and application

technical field

The invention belongs to the field of biotechnology, relates to a modified human lactoferrin gene suitable for expression in silk glands of silkworm, and also relates to an expression system and application for expressing the human lactoferrin gene.

Background technique

Since the beginning of the 21st century, with the increasing demand for functional proteins for various purposes such as medical, medicinal, edible, beauty, and health care, the extraction and production of natural sources of protein have been unable to meet the rapidly growing market demand. . Establish and improve various efficient prokaryotic and eukaryotic expression systems, using Escherichia coli, yeast, insect cells, mammalian cells, insects and mammals as host bioreactors, is the realization of low-cost, large-scale production with biological activity The effective and sustainable method of recombining exogenous proteins has become a research hotspot in the world today. Using Chinese hamster ovary cells as bioreactors to produce foreign proteins is the most standard expression mode at present, but its operating cost and environmental requirements are extremely harsh, which seriously limits its large-scale application. In order to establish a low-cost, large-scale, safe and sustainable biofactory for the efficient production of exogenous proteins, since 2000, researchers have begun to use genetically modified organisms, such as mammals, birds, insects and plant organs as bioreactors to produce recombinant foreign proteins.

Silkworm is known for its silk secretion, and it is one of the earliest economic animals (insects) that have been completely domesticated and utilized by humans. The silk gland of the silkworm is the organ that synthesizes and secretes silk protein, and is the biological basis of the entire silk industry. After thousands of years of artificial domestication, the silk glands of the silkworm have super-strong protein synthesis and secretion capabilities. A silkworm weighing about 5g can synthesize and secrete about 0.5g of silk protein, which is the most known insect. Silk protein is mainly composed of silk fibroin (fibroin) and sericin (sericin) wrapped in its outer layer: silk fibroin is the main body of silk, accounting for about 75%, and is synthesized by the posterior silk glands of silkworms, including fib- The three main components of H chain, fib-L chain and P25 are all insoluble in water; the rest is sericin, accounting for about 25%, which is synthesized by the middle silk gland of silkworm, including sericin 1 ( Sericin 1), sericin 2 ( Sericin 2 ) and sericin 3 ( Sericin 3 ) three main components, of which sericin 1 has the highest protein content, and both are soluble in water. With the development of modern molecular biology and transgenic technology, silkworm silk glands can efficiently synthesize and secrete silk proteins, as well as post-translational proteins such as glycosylation and methylation, which are extremely important to maintain the activity of exogenous proteins. The characteristics of modification and processing ability, low feeding cost, factory production, and safety for humans and animals make it an ideal bioreactor model, which has attracted the attention of researchers from all over the world and is competing for development and utilization.

In 2000, Tamura et al. used pBac transposon to mediate microinjection of silkworm eggs and obtained a stable genetic transgenic silkworm; in 2003, Xia Qingyou et al. completed the silkworm genome project. Encoding genes such as silk fibroin heavy chain (FibH chain) gene, silk fibroin light chain (FibL chain) gene, sericin1 (Sericin1) gene, sericin2 (Sericin2) gene, sericin3 (Sericin3) gene and P25 gene, etc. The regulatory elements of the promoter were identified and cloned. At the same time, the laboratory optimized the expression system in the early stage, and obtained an efficient transgenic silkworm sericin 1 expression system. These basic research results make it possible to use the transgenic silkworm tissue-specific expression system to produce recombinant exogenous proteins on a large scale in silk glands. In recent years, at home and abroad, using pBac transposon-mediated transgenic technology and silkworm tissue-specific promoter elements have tried to express a number of exogenous proteins in silk glands, including: the expression of silk fibroin heavy chain fusion in the posterior silk gland. EGFP (Zhao et al. 2010), feline interferon (Kurihara et al. 2007), spider silk traction protein (Zhu et al. 2010), partial peptides of human type III collagen (Tomita et al. 2003), enhanced Red fluorescent protein (Tomita et al. 2003), hydroxyproline collagen partial peptides fused to silk fibroin light chain (Adachi et al. 2006), fibroblast growth factor (Hino et al. 2006), enhanced green fluorescence Protein (Shimizu et al. 2007), partial collagen peptides (Yanagisawa et al. 2007), and red fluorescent protein fused to P25 (Royer et al. 2005), etc.; human serum albumin (Ogawa et al. 2005) was expressed in the middle silk gland. et al. 2007), enhanced green fluorescent protein (Tomita et al. 2007), murine monoclonal antibody (Iizuka et al. 2009), human collagen alpha chain gene (Adachi et al. 2010), and soluble GM-Csf Body alpha (Urano et al. 2010) et al. Based on the above research results at home and abroad, it is shown that the use of silk glands as bioreactors to produce exogenous proteins with high added value not only has broad market prospects, but also can break the barriers that silkworms can only be used as traditional industries. The development of new industries provides basic technical system guarantee.

Human lactoferrin is a single-chain glycoprotein composed of 716 amino acids with a molecular weight of about 78kDa. Human lactoferrin can transport and store iron and is a member of the transferrin family. Lactoferrin has many biological activities such as broad-spectrum antibacterial, antiviral, anti-inflammatory, inhibiting tumor cell growth and regulating the body's immune response. In addition, lactoferrin is closely related to the promotion of larval digestive tract development and iron absorption. At present, the application of lactoferrin in the market mainly focuses on adding it to milk powder as a nutritional fortifier to enhance the immunity of infants and young children. At present, the commercial production of human lactoferrin is mainly achieved through the separation and purification of whey. However, with the vigorous consumption of lactoferrin, the traditional separation and purification methods are difficult to meet the market demand. Traditional isolation methods are not only very expensive, but also carry the risk of spreading diseases, such as hepatitis and HIV. Regulatory authorities have also called on pharmaceutical companies to use non-animal-derived whey for the production of lactoferrin, which can effectively control the risk of infection by disease agents. Therefore, the use of gene recombination technology to produce human lactoferrin can not only effectively control the risk of disease transmission, but also can be used as a substitute for whey-derived human lactoferrin, which has a huge market development prospect. In 2009, Wang Zhenxing published a study on the expression of recombinant human lactoferrin using the silk gland bioreactor of the silkworm, but this method can only express a small amount of protein in the silk gland in the middle of the silkworm, and the expression of recombinant human lactoferrin is almost detected in silk. .

Therefore, it is urgent to develop a transgenic silkworm material with industrial value for high-efficiency and large-scale production of human lactoferrin recombinant protein, and to explore the feasibility of the practical application of the silk gland bioreactor of the transgenic silkworm.

SUMMARY OF THE INVENTION

In view of this, the object of the present invention is to provide a modified human lactoferrin gene suitable for expression in the silk gland of the silkworm; the second object of the present invention is to provide the modified human lactoferrin gene suitable for expression in the silk gland of the silkworm. The third purpose of the present invention is to provide the application of the expression system in the expression of recombinant human lactoferrin in the silk gland of the silkworm; the fourth purpose of the present invention is to provide the expression system of the recombinant human milk expressed in the silk gland of the silkworm by using the expression system ferritin method.

To achieve the above object, the present invention provides the following technical solutions:

1. A modified human lactoferrin gene suitable for expression in silk glands of Bombyx mori, the modified human lactoferrin gene is shown in SEQ ID NO.1.

2. An expression system comprising the modified human lactoferrin gene according to claim 1 or 2, which is suitable for expression in the silk gland of Bombyx mori.

Preferably, the expression system contains the sequence-linked enhancer hr3, the promoter of the secreted sericin 1 gene, the modified human lactoferrin gene and the terminator of the sericin 1 gene.

Preferably, the expression system further contains a fluorescent selection marker gene expression cassette, and the fluorescent selection marker gene expression cassette is located upstream of the enhancer hr3.

3. The application of the expression system in the expression of recombinant human lactoferrin in silk glands of silkworm.

4, utilize the described expression system to express the method for recombinant human lactoferrin in silk gland of silkworm, comprise the steps: described expression system is transformed to relieve diapause silkworm silkworm eggs, after sealing with nontoxic glue, through formaldehyde steam sterilization, hatch, Screening of transgenic positive moth circles, the positive transgenic silkworm cocoons were taken, pulverized into powder in liquid nitrogen, then dissolved in a buffer containing 50 mM Tris-Cl, 8 M Urea, pH 8.0, water bathed at 80 °C for 40 min, and the supernatant was collected by centrifugation. After purification, recombinant human lactoferrin was obtained.

Preferably, the concentration of the cocoon shell powder dissolved in the buffer is 20 mg/mL.

Preferably, the specific method of purification is: passing the supernatant through a His Trap ^TM FF crude affinity chromatography column, eluting with an imidazole eluent having an imidazole concentration of 0-500 mM, and using the imidazole eluent at pH 7.0 -7.5. The concentration is 8M Urea solution, and the eluate is collected. The eluate is the purified recombinant human lactoferrin.

The beneficial effects of the present invention are as follows: the present invention obtains a human lactoferrin gene sequence suitable for codon preference of silkworm by optimizing the coding sequence of human lactoferrin, and combines the optimized human lactoferrin gene sequence with the gene containing secreted sericin 1 The promoter and terminator constitute the expression frame, and the enhancer hr3 is used to enhance expression, fluorescent marker gene screening and piggyBac transposable arm to construct a high-efficiency expression system. Recombinant human lactoferrin, which is similar to ferritin, provides the possibility for efficient large-scale production of human lactoferrin recombinant protein. The produced recombinant human lactoferrin can replace blood-derived human lactoferrin and can effectively control the risk of disease transmission. Has huge market development prospects.

Description of drawings

In order to make the purpose, technical solutions and beneficial effects of the present invention clearer, the present invention provides the following drawings for description:

Figure 1. Structure diagram of transgenic expression vector (3xp3RFP represents transgenic fluorescent selectable marker gene; hr3CQ represents enhancer hr3; Ser1 represents secreted sericin 1 gene promoter; rhLF represents the coding sequence of human lactoferrin gene that has been codon-optimized; Ser1PA denotes the terminator of the sericin 1 gene; ITR denotes the piggyBac transposable arm sequence).

Figure 2 is a graph of transgenic fluorescence screening (A: moth white light image; B: moth fluorescence image).

Figure 3 is the SDS-Page electrophoresis image of sgrhLF silkworm cocoon protein (1-65: cocoon protein of 65 transgenic positive individuals; WT: normal cocoon protein).

Figure 4 is the detection of rhLF in sgrhLF silkworm cocoon shell protein (A: SDS-Page electrophoresis image; B: Western blot image).

Figure 5 shows the quantitative detection of rhLF sericin solution and purification (A: SDS-PAGE electrophoresis; B: Western blot detection).

Figure 6 is the secondary structure detection of recombinant rhLF protein.

Figure 7 shows that rhLF reduces LPS-induced inflammation in cells (A: TNF-α content; B: NO content; C: Western blot detection results of iNOs production in cells; D: relative expression of iNOs).

Figure 8 shows the results of the detection of the antibacterial activity of recombinant human lactoferrin.

Detailed ways

The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

The materials used in the examples of the present invention are as follows: the silkworm variety Dazao (P50) to be tested is preserved by this laboratory. The larvae were reared on artificial diet in an artificial climate box at 25°C. Mouse peritoneal macrophages raw264.7 were preserved in the laboratory and cultured in DMEM medium containing 10% (v/v) fetal bovine serum (FBS, Gibco) at 37°C, 5% CO ₂ . Plasmid vectors pSLfa1180fa, pBac{3xp3EGFPaf}, pBac{3xp3DsRedaf} are preserved by our laboratory.

Preparation of main reagents and solutions: For the conventional medium and reagent buffers used in the molecular cloning process, please refer to the "Molecular Cloning Experiment Guide Third Edition Translation" and the "TaKaRa Product Catalog" in the chapter on the preparation of laboratory routine reagents (S1- S11) to configure. DNA polymerase Ex-Taq, LA-Taq Kit, conventional restriction enzymes, alkaline phosphatase, sequencing cloning vector pMD19-T simple vector Kit, DNA Ligation Kit Ver.2.0, fluorescence quantitative PCR kit SYBRpremix Ex TaqTM are all Available from TaKaRa Company. E. coli competent cells Trans1-T1 were used for transformation, and the conventional plasmid DNA extraction kit, Easypure Plasmid MiniPrep Kit, was purchased from Quanzhijin Company. Agarose gel DNA recovery kit Gel Extraction Mini Kit (50) was purchased from Huashun Biotechnology Company. The ultrapure plasmid extraction kit for transgene injection, QIA prep Spin Miniprep Kit (50), was purchased from QIAGEN. Total RNA Kit II (50) was purchased from Omega Bio-Tec. Human lactoferrin polyclonal antibody anti-rhLF antibody and recombinant human lactoferrin standard rhLF-std were purchased from Sigma Company, nitric oxide synthase (iNOs) polyclonal antibody anti-iNOs antibody was purchased from abcam, NO detection reagent The kit was purchased from Biyuntian Company, and the TNF-α detection kit was purchased from R&D systems Company.

Example 1, the synthesis of the modified human lactoferrin gene

The amino acid sequence of mature peptide of human lactoferin (hLF, GenBank: M93150.1) was downloaded from NCBI, and the coding sequence was optimized and designed according to the codon usage preference of Bombyx mori. The specific sequence is shown in positions 7-2106 of SEQ ID NO. As shown, the amino acid sequence is shown in the 1st to 700th positions of SEQ ID NO.2, and the gene sequence was synthesized by the company. The amino acid sequence of human lactoferrin (hLF) was fused to the C-terminus of 6 histidine amino acids to form hLF-his6. The two ends of the synthesized two recombinant protein gene sequences are respectively connected with BamHI and NotI restriction sites, as shown in SEQ ID NO. 1, and the encoded amino acid sequence is shown in SEQ ID NO. 2.

Example 2. Construction of transgenic expression vector

The commercially synthesized hLF-his6 gene coding sequence was constructed into psl1180[hr3Pser1spRedSer1PA] through BamHI and NotI restriction sites to form psl1180[hr3CQSer1sphLFSer1], the nucleotide sequence is shown in SEQ ID NO.3, and then constructed through the AscI site To the AscI site of the pBac{3xp3EGFPaf} vector, the transgenic expression vector phShLFSer is formed, and the structure of the vector is shown in Figure 1.

Example 3. Microinjection and Fluorescence Screening

The transgenic expression vector phShLFSer1 and the auxiliary vector pHA3PIG plasmid were extracted by QIAGEN Plasimd Mini Kit plasmid extraction kit, the concentration of each plasmid was diluted to 400ng/μl, and mixed with the auxiliary vector pHA3PIG plasmid at a molar ratio of 1:1. The mixed plasmids were injected into the diapause-released Dazao early embryos (2 to 5 hours after spawning), then the injection holes were sealed with non-toxic glue, sterilized with 35% formaldehyde steam for 5 minutes, and then placed at 25°C, Incubate in an environment with a relative humidity of 85%. The hatched larvae (G0 generation) are raised with artificial diet. After reaching the adult, self-crossing or backcrossing is carried out to produce seeds. The obtained G1 generation silkworm eggs (7th day) are observed in a macroscopic stereoscopic fluorescence microscope. (Olypus MVX10, Japan), the red fluorescence was observed using excitation light with a wavelength of 510-550 nm, and the transgenic-positive moth circles that specifically excited red fluorescence in the eyes or nerves were screened and named sghLF, as shown in Figure 2. The fluorescence screening statistics of transgenic silkworms are shown in Table 1.

Table 1. Transgene Screening Statistics

The results showed that a total of 12 positive moth rings were screened from 43 G1 moth rings of sgrhLF, and the positive rate was 27.90%.

Example 4. SDS-Page and Western blot detection

Extraction and detection method of recombinant protein in total protein of cocoon shell: pulverize cocoon shell into powder in liquid nitrogen, dissolve in 50mM Tris-Cl, pH 8.0, 8M Urea buffer according to the concentration of cocoon shell of 20mg/ml, 80 ℃ water bath for 40min, then centrifuged to collect the supernatant, centrifugation conditions: 13500×rpm, 25℃, 15min. The extracted cocoon shell total protein was detected by 12% SDS-Page electrophoresis and stained with Coomassie brilliant blue. After the extracted total protein was separated by 12% SDS-Page gel electrophoresis, the protein in the SDS-Page gel was transferred to PVDF membrane by electrotransfer method. PVDF membranes were blocked in TBST buffer containing 5% nonfat dry milk overnight at 4°C. Before immunohybridization, the PVDF membrane was washed three times with TBST at room temperature for 5 min each time. Anti-rhLF (Sigma) primary antibody hybridization solution was prepared in TBST containing 5% nonfat milk powder at a dilution ratio of 1000 times. The PVDF membrane was immersed in the hybridization solution and incubated with shaking at room temperature for 2 h, and the membrane was washed 5 times with TBST for 10 min each time. The hybridization solution of HRP-labeled goat anti-rabbit secondary antibody (purchased from Biyuntian Co., Ltd.) was prepared with TBST at a dilution ratio of 20,000 times. The PVDF membrane washed with TBST was immersed in the secondary antibody hybridization solution and incubated at room temperature with shaking for 2 hours. The membrane was washed 5 times with TBST. , 10min each time. The cleaned PVDF membrane was placed on a clean plastic wrap, and ECL chromogenic solution (Amersham Biosciences) was evenly dropped on the PDVF membrane, incubated at room temperature for 5 min in the dark, and exposed and imaged using a ChemiscopeSeries (Clinx science instruments).

The SDS-Page detection results are shown in Figure 3. The results show that the transgenic cocoon shell protein has a differential band above the 70kDa molecular marker, which is consistent with the theoretical molecular weight of the rhLF protein, and the differential band is in the cocoon shell derived from different transgenic individuals. The content of total protein varies significantly. In order to further identify this specific band as rhLF, Western blot was performed on the cocoon protein numbered 53-65, and the results are shown in Figure 4. The results showed that the differential band of transgenic cocoon shell protein above the 70kDa molecular marker could react specifically with the rhLF antibody, which confirmed that the differential band was the rhLF protein specifically expressed by the transgenic silkworm.

Through grayscale comparison with the rhLF standard, it was calculated that the content of rhLF protein in 1 g of silk was about 10.27 mg, that is, rhLF accounted for 0.93% in rhLF transgenic silkworm cocoons (Figure 5). The above results show that the Ser1 expression system established in this study can efficiently recombinantly produce human lactoferrin and secrete it into Bombyx mori silk. In addition, the contents of recombinant proteins in different positive moth ring-derived individuals were significantly different, suggesting that the expression of transgenic rhLF gene in Bombyx mori silk gland cells was affected by a strong chromosomal location effect.

However, using Wang Zhenxing's method only detected 192 μg/ml in silk glands, and it was calculated that the content of rhLF protein in 1 g of silk glands was about 2.4 mg, while almost no rhLF protein was detected in silk.

Example 5. Separation and purification of rhLF from silkworm cocoons and detection

1 g of hLF transgenic silkworm cocoons were minced and placed in 50 mM Tris-Cl, pH 8.0, 8 M Urea buffer, 80 °C water bath for 40 min, 13500 rpm, 25 °C centrifugation for 15 min to collect the supernatant. The supernatant was first passed through a His Trap ^TM FF crude affinity chromatography column (GE healthcare) to bind rhLF to nickel ions in the column, and then prepared with different concentrations of imidazole eluents (8MUrea, pH 7.0-7.5, Imidazole concentrations of 0, 20, 50, 100, 500 mM) were eluted and collected. After the samples were collected, 12% SDS-PAGE and western blot were performed to detect their purification and yield. The results showed that the transgenic silkworm cocoon extracted protein with a differential protein band above the 70kDa molecular marker, and the differential protein band could specifically react with the antibody of rhLF. rhLF dissolves out. Further analysis of the SDS-PAGE and Western blot results of the purified rhLF showed that rhLF protein with a purity of about 90% could be obtained. 1 g of silk cocoons could purify 9.21 mg of rhLF, and the purification yield of rhLF was as high as 89.25% (Figure 5).

(1) Recombinant protein secondary structure detection

The purified rhLF sample was dissolved in deionized water, and the CD spectrum of rhLF between 190-250 nm was measured at a concentration of 0.5 mg/mL. The results are shown in Figure 6. The results showed that at CD: 190-250nm, the rhLF expressed by the transgenic silkworm had a similar absorption peak with the commercial rhLF standard protein, and by calculation, the silkworm expressed rhLF and the commercial rhLF standard α -helix, β -sheet , β -turn and random coil and other protein secondary structures are relatively consistent (Table 2). Therefore, we speculate that rhLF expressed by transgenic silkworm should have its biological function. This result indicates that the Ser1 expression system established in this study can achieve high-efficiency recombinant expression and isolation and purification of foreign proteins.

Table 2. Secondary structure analysis of recombinant rhLF protein

sample name <i>α</i>-helix (%) <i>β</i>-fold (%) <i>β</i>-angle (%) random coil (%) rhLF-std 52.60 10.35 14.90 22.50 rhLF 51.20 10.90 15.30 22.90

(2) Detection of anti-inflammatory activity of recombinant human lactoferrin

The purified rhLF samples were dissolved in deionized water for use. The raw264.7 cells were resuspended and inoculated into a 96-well cell culture plate at a density of 5 x 10 ⁴ cells/well. After culturing for 12 hours, the cells adhered to the wall. Bacterial lipopolysaccharide (LPS, 50ng/μl) and different concentrations of rhLF (final concentration) were added. 0μg/ml, 10μg/ml, 20μg/ml, 30μg/ml) for 12h, and rhLFstd was the positive control; after 12h, the cell culture medium kit was taken to measure the concentrations of NO and TNF-α, and the cells were washed twice with ice PBS and lysed. The cells were lysed in solution, and the expression of iNOs in the cells was determined by Western blotting. The results are shown in Figure 7. The results showed that LPS could induce raw264.7 cells to produce NO, TNF-α and other inflammatory factors and overexpression of iNOs. The GRRRR and RKVRGPP sequences at the N-terminal of human lactoferrin can bind to LPS, thereby reducing the production of inflammatory factors by LPS-induced cells. The anti-inflammatory activity detection of purified rhLF showed that 10μg/ml rhLF could significantly reduce LPS-induced TNF-α and NO production in raw264.7 cells, and with the increase of concentration, the inhibition rate increased; Western blot detection The results of the production of iNOs by the cells showed that when the concentration of rhLF was 20 μg/ml, the production of iNOs in the cells was significantly reduced compared with the blank control group. Using the commercial rhLF standard as a positive control, the rhLF and rhLF standards expressed in the silk glands of Bombyx mori reduced the production of iNOs in the cells. Inflammatory factor production effect is comparable. The above experimental results showed that recombinant human lactoferrin (rhLF) expressed in silk glands of Bombyx mori has anti-inflammatory activity.

(3) Detection of antibacterial activity of recombinant human lactoferrin

The purified rhLF samples were dissolved in deionized water for use. E. coli was activated with LB medium and inoculated into a 96-well culture plate, 100 μl/well, added with different concentrations of rhLF (concentrations of 0.5 mg/ml, 1 mg/ml, 2 mg/ml) and incubated with E. coli, at 600 nm every 1 h. The absorbance value was measured for 12 hours continuously, and the results are shown in Figure 8. The results showed that rhLF was dose-dependent and time-dependent on the growth of E. coli. Human lactoferrin can competitively bind to the iron ions required for the growth of E. coli, thereby inhibiting the growth of E. coli. The results of purified rhLF inhibiting Escherichia coli showed that rhLF had an inhibitory effect on the growth of Escherichia coli from the 3rd hour, and with the increase of time, the inhibition rate increased, and the bacterial growth tended to be flat at 8h; in addition, high concentration of rhLF (2mg /ml) inhibition effect The lower concentration (0.5mg/ml) of rhLF inhibited significantly; at 12h, the inhibition rate of 2mg/ml rhLF reached the highest 29.85%.

To sum up, the present invention utilizes the established Ser1 expression system hSRSE to recombinantly express human lactoferrin (rhLF). The gene encoding human lactoferrin (rhLF) was artificially designed and synthesized according to the preference of codon usage in the silkworm genome, and the transgenic expression vector phSrhLFSer1 was constructed. By microinjecting silkworm embryos, we established a transgenic silkworm line sgrhLF, in which 7 lines without positive moth ring origin were obtained by sgrhLF screening.

Using SDS-PAGE and Western blot detection methods, the high expression of rhLF protein was detected in the cocoons of sgrhLF silkworm. Compared with the rhLF standard, the rhLF protein content in sgrhLF cocoon silk was calculated to be 10.27 mg/g. The results show that the expression efficiency of the hSRSE expression system established by systematic optimization has reached the level of the expression system currently used abroad. On this basis, we used urea to separate and purify sgrhLF silkworm silk protein. At this stage, rhLF protein with a purity of 90% can be obtained by one-step purification through His Trap ^TM FF crude affinity chromatography column. With further optimization, the purity of the recombinant protein will be further improved. Subsequently, we performed CD spectrum scanning on the secondary spatial structure of the purified recombinant protein. The results showed that the rhLF protein and the commercial rhLF protein had the same secondary structure. Has the same biological activity as commercial rhLF.

Therefore, through the recombinant expression, isolation, purification and activity identification of the rhLF target exogenous protein, it was preliminarily confirmed that the previously established Ser1 expression system hSRSE can realize the efficient secretion and expression of the exogenous protein in the silk gland of the transgenic silkworm, and produce biologically Active recombinant protein. The results indicated that the hSRSE expression system could be the basic technical system for the practical process of silk gland biological response in Bombyx mori.

Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should Various changes may be made in details without departing from the scope of the invention as defined by the claims.

sequence listing

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<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala Val Ser Gln Pro

1 5 10 15

Glu Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg

20 25 30

Gly Pro Pro Val Ser Cys Ile Lys Arg Asp Ser Pro Ile Gln Cys Ile

35 40 45

Gln Ala Ile Ala Glu Asn Arg Ala Asp Ala Val Thr Leu Asp Gly Gly

50 55 60

Phe Ile Tyr Glu Ala Gly Leu Ala Pro Tyr Lys Leu Arg Pro Val Ala

65 70 75 80

Ala Glu Val Tyr Gly Thr Glu Arg Gln Pro Arg Thr His Tyr Tyr Ala

85 90 95

Val Ala Val Val Lys Lys Gly Gly Ser Phe Gln Leu Asn Glu Leu Gln

100 105 110

Gly Leu Lys Ser Cys His Thr Gly Leu Arg Arg Thr Ala Gly Trp Asn

115 120 125

Val Pro Thr Gly Thr Leu Arg Pro Phe Leu Asn Trp Thr Gly Pro Pro

130 135 140

Glu Pro Ile Glu Ala Ala Val Ala Arg Phe Phe Ser Ala Ser Cys Val

145 150 155 160

Pro Gly Ala Asp Lys Gly Gln Phe Pro Asn Leu Cys Arg Leu Cys Ala

165 170 175

Gly Thr Gly Glu Asn Lys Cys Ala Phe Ser Ser Gln Glu Pro Tyr Phe

180 185 190

Ser Tyr Ser Gly Ala Phe Lys Cys Leu Arg Asp Gly Ala Gly Asp Val

195 200 205

Ala Phe Ile Arg Glu Ser Thr Val Phe Glu Asp Leu Ser Asp Glu Ala

210 215 220

Glu Arg Asp Glu Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg Lys Pro

225 230 235 240

Val Asp Lys Phe Lys Asp Cys His Leu Ala Arg Val Pro Ser His Ala

245 250 255

Val Val Ala Arg Ser Val Asn Gly Lys Glu Asp Ala Ile Trp Asn Leu

260 265 270

Leu Arg Gln Ala Gln Glu Lys Phe Gly Lys Asp Lys Ser Pro Lys Phe

275 280 285

Gln Leu Phe Gly Ser Pro Ser Gly Gln Lys Asp Leu Leu Phe Lys Asp

290 295 300

Ser Ala Ile Gly Phe Ser Arg Val Pro Pro Arg Ile Asp Ser Gly Leu

305 310 315 320

Tyr Leu Gly Ser Gly Tyr Phe Thr Ala Ile Gln Asn Leu Arg Lys Ser

325 330 335

Glu Glu Glu Val Ala Ala Arg Arg Ala Arg Val Val Trp Cys Ala Val

340 345 350

Gly Glu Gln Glu Leu Arg Lys Cys Asn Gln Trp Ser Gly Leu Ser Glu

355 360 365

Gly Ser Val Thr Cys Ser Ser Ala Ser Thr Thr Glu Asp Cys Ile Ala

370 375 380

Leu Val Leu Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Tyr

385 390 395 400

Val Tyr Thr Ala Cys Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn

405 410 415

Tyr Lys Ser Gln Gln Ser Ser Asp Pro Asp Pro Asn Cys Val Asp Arg

420 425 430

Pro Val Glu Gly Tyr Leu Ala Val Ala Val Val Arg Arg Ser Asp Thr

435 440 445

Ser Leu Thr Trp Asn Ser Val Lys Gly Lys Lys Ser Cys His Thr Ala

450 455 460

Val Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Leu Phe Asn

465 470 475 480

Gln Thr Gly Ser Cys Lys Phe Asp Glu Tyr Phe Ser Gln Ser Cys Ala

485 490 495

Pro Gly Ser Asp Pro Arg Ser Asn Leu Cys Ala Leu Cys Ile Gly Asp

500 505 510

Glu Gln Gly Glu Asn Lys Cys Val Pro Asn Ser Asn Glu Arg Tyr Tyr

515 520 525

Gly Tyr Thr Gly Ala Phe Arg Cys Leu Ala Glu Asn Ala Gly Asp Val

530 535 540

Ala Phe Val Lys Asp Val Thr Val Leu Gln Asn Thr Asp Gly Asn Asn

545 550 555 560

Asn Glu Ala Trp Ala Lys Asp Leu Lys Leu Ala Asp Phe Ala Leu Leu

565 570 575

Cys Leu Asp Gly Lys Arg Lys Pro Val Thr Glu Ala Arg Ser Cys His

580 585 590

Leu Ala Met Ala Pro Asn His Ala Val Val Ser Arg Met Asp Lys Val

595 600 605

Glu Arg Leu Lys Gln Val Leu Leu His Gln Gln Ala Lys Phe Gly Arg

610 615 620

Asn Gly Ser Asp Cys Pro Asp Lys Phe Cys Leu Phe Gln Ser Glu Thr

625 630 635 640

Lys Asn Leu Leu Phe Asn Asp Asn Thr Glu Cys Leu Ala Arg Leu His

645 650 655

Gly Lys Thr Thr Tyr Glu Lys Tyr Leu Gly Pro Gln Tyr Val Ala Gly

660 665 670

Ile Thr Asn Leu Lys Lys Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys

675 680 685

Glu Phe Leu Arg Lys Glu Asn Leu Tyr Phe Gln Ser His His His

690 695 700

His His

705

<210> 3

<211> 4157

<212> DNA

<213> Artificial Sequence

<400> 3

ccatggcagc gtcgtgaaaa gaggcaatga caaatacaaa acgacgtatg agcagacccg 60

tcgccaagac gggtctacct ctaagatgat gtcatttgtt ttttaaaact aactcgcttt 120

acgagtagaa ttctacgtgt aaaacataat caagagatga tgtcatttgt ttttcaaaac 180

caaactcgct ttacgagtag aattctacgt gtaaaacaca atcaaaagat gatgtcattc 240

gtttttcaaa accgaattta agaaatgatg tcatttgttt ttcaaaacca aactcgcttt 300

acgagcagaa ttctacgtgt aaaacacaat caagagatga tgtcatttgt ttttcaaaac 360

tgaatgatgt catttgtttt tcaaaactaa acttgctttg cgagtagaat tctacgtgta 420

aaacacagtc aagagatgat gtcatttgtt tttcaaaact gaaccggctt tacgagtaga 480

attctacttg taaaacataa tcaagagatg atgtcatttg tttttcaaaa ctgaactggc 540

tttacgagta gaattctacg tgtaaaacat aatcaagaga tgatgtcatc attaaactga 600

tgtcatttta tacacgattg ttaacatgtt taataatgac taatttgttt ttccaaatta 660

aactcgcttt acgagtagaa ttctacttgt aacgcacgat taagtatgaa tcataagctg 720

atgtcatttg ttttcgacat aaaatgttta tacaatggaa tcttcttgta aattatccaa 780

ataatataat ttatccgatt ctacgttaca tttaaattcg ttgttatcgt acaattcttc 840

aggacacgcc atgtattggt catttttagc gtgcaaccaa cgattgtatt tgacgccgtc 900

gttggattgc gtgttcaggt tggcgtacac gtgactgggc acggcttctt tttccatggg 960

acgtcgacga aaacagcaca cacactacat accatgtatt tgacgcacac acgcatgtat 1020

actatttatt gtcaaacttt tgttcttgac gtctgtgttc aaactgagaa tagattaaat 1080

attgtttgtc tttattaata ttttttaata gtgtagtctt ggcgaaattt gtgattataa 1140

aagtataaaa tacaatcata atagtgtacg aacttacaat tccaattaat tatagtcgaa 1200

tttcgactac tgcgggacct ctagtattaa taattctctt taaaaaaaaa cagagcatca 1260

aatactgcac aaatgtcaag cgggtctcaa cgagccatga ataaattaga aatcaattaa 1320

taacataaaa taggcaaaca aaataaaacc atttacatag agaacgtttg ttgaacaaaa 1380

acaataactt gtatacattg tttgcacaaa tgtttgaagc gaaaatttat tactctctac 1440

gtaagcttga tcaaacttcg ttttcgtata aaacgcgttg gcccaaccac tttggcatag 1500

tcgtcttatc atcgggtctc taaggatcaa gcgatccaaa gaccgccaac atgcgtttcg 1560

ttctgtgctg cactttgatt gcgttggctg cgctcagcgt aaaagccttc ggtcaccacc 1620

ccggcaatcg agatacagga tccatgggca gaagaagaag atctgtccaa tggtgcgctg 1680

tgagccagcc ggaagccacc aaatgtttcc aatggcagag aaatatgaga aaggttagag 1740

gtcctccagt ctcttgcata aaaagagata gccccatcca atgtattcag gctatagccg 1800

aaaacagagc tgacgccgtg acattggatg gtggattcat ctacgaagct ggtctcgccc 1860

cgtacaaact gagacccgtt gctgccgaag tctacggaac cgaaagacaa ccgagaacac 1920

actactacgc tgtggccgtg gttaaaaagg gcggttcttt ccaattgaat gaactccagg 1980

gtctgaagag ctgccacacc ggtctgagaa gaacagctgg atggaatgtt cctactggca 2040

ccttgagacc attcctcaac tggacaggac cgcccgaacc tattgaagct gccgttgcta 2100

gattcttctc agcctcttgc gtccctggag ctgacaaggg ccaattccca aatttgtgca 2160

gactctgtgc tggtactgga gaaaacaaat gcgccttctc atctcaggaa ccatacttca 2220

gctactccgg agctttcaag tgtttgagag acggcgctgg tgacgtcgcc ttcataagag 2280

aatccaccgt gttcgaagac ctctcggatg aagctgaaag agacgaatac gaactgttgt 2340

gcccggataa cacaagaaaa cccgttgaca aattcaagga ttgtcacctc gctagagtgc 2400

caagtcacgc tgtcgtggcc agatcagtta atggtaaaga agatgctatc tggaacctcc 2460

tgagacaagc ccaggaaaaa ttcggaaaag acaagagccc gaagttccaa ctgttcggtt 2520

cgcccagtgg acagaaagac ttgctcttca aggatagtgc tatcggattc tcaagagtcc 2580

ctccaagaat tgattccggc ttgtacctcg gatcgggcta cttcacagcc atccagaatt 2640

tgagaaaatc agaagaagaa gttgctgcca gaagagctag agttgtctgg tgcgccgtcg 2700

gtgaacaaga actgagaaag tgtaaccagt ggtcgggctt gagtgaaggt tcagtgactt 2760

gcagctccgc ttctacaact gaagactgta ttgccctggt tttgaaaggc gaagctgacg 2820

ccatgagcct ggatggaggc tacgtctaca ccgcttgcaa gtgtggtctg gttcctgtct 2880

tggccgaaaa ttacaaatct caacagtcgt cagaccctga ccctaactgc gtggatagac 2940

cagttgaagg atacttggct gtggccgtgg ttagaagatc tgacacaagc ctcacttgga 3000

acagtgtcaa aggcaaaaag tcatgtcaca ctgctgtgga cagaaccgcc ggttggaata 3060

ttccgatggg actgttgttc aaccaaactg gctcctgcaa gttcgacgaa tacttctcac 3120

agtcttgtgc tcctggttcc gatccaagat cgaatctgtg cgccttgtgt ataggagacg 3180

aacaaggcga aaacaaatgc gtccccaatt cgaacgaaag atactacggt tacaccggag 3240

ctttcagatg tctcgctgaa aatgccggag acgtcgcctt cgtgaaggat gtgacagttc 3300

tgcagaacac tgatggcaac aataacgaag cttgggccaa agacctcaag ctggctgatt 3360

tcgccctcct gtgcctggac ggaaaaagaa agcctgtgac tgaagctaga agttgtcact 3420

tggctatggc cccaaaccac gccgtcgtgt caagaatgga taaagtggaa agactgaagc 3480

aagttttgct ccaccaacag gctaaattcg gcagaaatgg ttccgactgc cctgataaat 3540

tctgtttgtt ccagtcggaa acaaagaacc tgttgttcaa tgacaacact gaatgcctcg 3600

ctagactgca cggcaaaacc acatacgaaa agtacctcgg cccgcaatac gttgctggta 3660

taactaatct gaaaaagtgc agcacctccc ccctcctgga agcctgtgaa ttcttgagaa 3720

aagaaaacct ctacttccag tcacaccacc accaccacca ctaagcggcc gctacaacta 3780

aacacgactt ggagtattcc ttgtagtgtt taagatttta aatcttactt aatgacttcg 3840

aacgatttta acgataactt tctctttgtt taactttaat cagcatacat aaaaagcccc 3900

ggttttgtat cgggaagaaa aaaaatgtaa ttgtgttgcc tagataataa acgtattatc 3960

aaagtgtgtg gttttccttt accaaagacc cctttaagat gggcctaatg ggcttaagtc 4020

gagtcctttc cgatgtgtta aatacacatt tattacactg atgcgtcgaa tgtacacttt 4080

taataggata gctccactaa aaattatttt atttatttaa tttgttgcac caaaactgat 4140

acattgacga aaagctt 4157

Claims (8)

1. The modified human lactoferrin gene suitable for expression in silk glands of silkworm is characterized in that: the modified human lactoferrin gene is shown in SEQ ID NO.1. 2. The expression vector containing the modified human lactoferrin gene according to claim 1, which is suitable for expression in the silk gland of Bombyx mori. 3 . The expression vector according to claim 2 , wherein the expression vector contains the sequence-linked enhancer hr3, the promoter of the secreted sericin 1 gene, the modified human lactoferrin gene and the termination of the sericin 1 gene. 4 . son. 4 . The expression vector according to claim 2 , wherein the expression vector further contains a fluorescent selection marker gene expression cassette, and the fluorescent selection marker gene expression cassette is located upstream of the enhancer hr3. 5 . 5. The application of the expression vector described in claim 3 or 4 in expressing recombinant human lactoferrin in silk gland of silkworm. 6. utilize the described expression vector of claim 3 or 4 to express the method for recombinant human lactoferrin in silk gland of silkworm, it is characterized in that, comprise the steps: described expression vector is transformed to relieve diapause silkworm eggs, use nontoxic glue After sealing, it was sterilized by formaldehyde steam, incubated, and screened for transgenic positive moth circles. The positive transgenic silkworm cocoons were taken, pulverized into powder in liquid nitrogen, and then dissolved in a buffer containing 50mM Tris-Cl, 8M Urea, pH 8.0, 80 The supernatant was collected by centrifugation in a water bath for 40 min, and purified to obtain recombinant human lactoferrin. 7 . The method according to claim 6 , wherein the concentration of the cocoon shell powder dissolved in the buffer is 20 mg/mL. 8 . 8. method according to claim 6, is characterized in that, the concrete method of described purification is: the supernatant liquid is crossed HisTrap ^TM FF crude affinity chromatography column, be the imidazole eluent of 0～500mM with imidazole concentration Elution, the imidazole eluate is prepared with a pH 7.0-7.5 and a concentration of 8M Urea solution, the eluate is collected, and the eluate is purified recombinant human lactoferrin.

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