Method for efficiently synthesizing recombinant human lactoferrin

Abstract

The present invention discloses a method for efficiently synthesizing recombinant human lactoferrin, comprising: transforming a target gene at least twice into Chlamydomonas reinhardtii microalgae to construct a microalgae engineering bacterium, wherein the target gene is a gene capable of expressing human lactoferrin; inoculating the microalgae engineering bacterium into a liquid culture medium and culturing under light at a temperature of 20 to 30°C, harvesting the algae liquid, and obtaining recombinant human lactoferrin from the algae liquid. The present invention transforms the human lactoferrin HLF gene at least twice in Chlamydomonas reinhardtii, introduces a human lactoferrin synthesis pathway, constructs an engineered algae strain with a high expression level, adjusts the culture medium formula on this basis, and cultivates under 5L fermentation amplification conditions, and finally achieves a maximum yield of human lactoferrin that is about 7 times higher than before the optimization of the culture conditions, reaching 1.1% of the cell dry weight.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a method for efficiently synthesizing recombinant human lactoferrin. Background Art

[0002] Lactoferrin, a multifunctional glycoprotein with about 700 amino acids and a molecular weight of 80 kDa, has high homology among species. Among them, human lactoferrin has a wide range of biological functions, including antiviral, antibacterial and antioxidant activities, regulating cell growth rate, and participating in innate / specific immune responses. It has broad application prospects in medicine, enzymes, food ingredients, nutritional additives and antibodies, industry, health care, research and daily life. However, the yield of natural lactoferrin is low, and the cost of separation and purification is high. Heterologous production of human lactoferrin through genetic engineering technology is an ideal way to solve the above problems. At present, recombinant human lactoferrin has been expressed in a series of organisms, including yeast, filamentous fungi and higher plants, but the expression effect is not ideal. It is urgent to develop an efficient human lactoferrin expression system and mass produce human lactoferrin. Summary of the invention

[0003] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0004] In view of the deficiencies of the prior art and actual needs, another object of the present invention is to provide a method for efficiently synthesizing recombinant human lactoferrin. The present invention synthesizes human lactoferrin by constructing a recombinant expression algae strain, and improves the amount of human lactoferrin synthesized by the recombinant algae strain through fermentation optimization.

[0005] To this end, the technical solution provided by the present invention is:

[0006] In a first aspect, a method for constructing an engineered microalgae strain comprises: transforming a target gene into Rhineland-Palatinate microalgae at least twice to construct an engineered microalgae strain, and each time the target gene is successfully expressed in the Rhineland-Palatinate microalgae, wherein the target gene is a gene capable of expressing human lactoferrin.

[0007] Preferably, in the method for constructing the microalgae engineering strain, the nucleotide sequence of the target gene is as shown in SEQ ID NO:2.

[0008] In a second aspect, a microalgae engineering strain is constructed by the following method: a target gene is transformed into Rhineland-White Microalgae at least twice to construct a microalgae engineering strain, and the target gene transformed into Rhineland-White Microalgae at least twice is successfully expressed, and the nucleotide sequence of the target gene is shown in SEQ ID NO:2.

[0009] In a third aspect, a method for efficiently synthesizing recombinant human lactoferrin comprises the following steps:

[0010] 1) The target gene is transformed into Rhineland-Palatinate microalgae at least twice to construct an engineered microalgae bacterium, and the target gene is successfully expressed each time it is transformed into Rhineland-Palatinate microalgae, wherein the target gene is a gene capable of expressing human lactoferrin;

[0011] 2) The microalgae engineered bacteria are inoculated into a liquid culture medium and cultured under light at a temperature of 20-30° C., the algae liquid is harvested, and the recombinant human lactoferrin is obtained from the algae liquid.

[0012] Preferably, in the method for efficiently synthesizing recombinant human lactoferrin, in step 2), the liquid culture medium is liquid TAP culture medium.

[0013] Preferably, in the method for efficiently synthesizing recombinant human lactoferrin, in step 2), the liquid culture medium is additionally added with Fe 2+ .

[0014] Preferably, in the method for efficiently synthesizing recombinant human lactoferrin, the liquid culture medium contains valine at a concentration of 8 mM.

[0015] Preferably, in the method for efficiently synthesizing recombinant human lactoferrin, in step 2), sodium acetate at a concentration of 2 to 10 g / L is additionally added to the liquid culture medium.

[0016] Preferably, in the method for efficiently synthesizing recombinant human lactoferrin, the TAP liquid culture medium is additionally supplemented with 0.3 mM Fe 2+ , 8 mM valine and 6 g / L sodium acetate.

[0017] Preferably, in the method for efficiently synthesizing recombinant human lactoferrin, in step 1), transforming the target gene into Chlamydomonas reinhardtii at least twice to construct an engineered microalgae bacteria comprises the following steps:

[0018] The gene sequence shown in SEQ ID NO: 2 was constructed into a Chlamydomonas reinhardtii expression vector, and then the Chlamydomonas reinhardtii expression vector was transferred into Chlamydomonas reinhardtii using a glass bead method, and the Chlamydomonas reinhardtii strain HLF6 with successful expression was obtained by screening;

[0019] The gene sequence shown in SEQ ID NO: 2 was constructed into another Chlamydomonas reinhardtii expression vector, and the other Chlamydomonas reinhardtii expression vector was transferred into the Chlamydomonas reinhardtii strain HLF6 and screened to obtain the Chlamydomonas reinhardtii strain HLF6-18 with successful expression.

[0020] Preferably, in the method for efficiently synthesizing recombinant human lactoferrin, the Chlamydomonas reinhardtii strain HLF6-18 in the logarithmic growth phase is inoculated into a fermentation tank. The culture medium contains 0.04-0.4 mM Fe² + , valine and 2-10 g / L sodium acetate in liquid TAP medium.

[0021] Preferably, in the method for efficiently synthesizing recombinant human lactoferrin, in step 2), the illumination culture time is 5 to 10 days, and the illumination intensity is 60 to 100 μmol / (m 2 More preferably, the illumination culture time is 7 days, and the illumination intensity is 80 μmol / (m 2 ·s).

[0022] The present invention has at least the following beneficial effects:

[0023] The present invention transforms the human lactoferrin HLF gene at least twice in Chlamydomonas reinhardtii UVM4, introduces the human lactoferrin synthesis pathway, constructs an engineered algae strain HLF6-18 with a high expression level, and adds Fe 2+ , valine and sodium acetate (as carbon source), and cultured under 5 L fermentation scale-up culture conditions. The maximum yield of human lactoferrin was finally increased by about 7 times compared with that before the optimization of culture conditions.

[0024] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the agarose gel electrophoresis detection result of PCR identification of Chlamydomonas reinhardtii that was first introduced into HLF in the example of the present invention.

[0026] Figure 2 1 is the mRNA expression of the human lactoferrin gene in different transgenic Chlamydomonas in the examples of the present invention.

[0027] Figure 3 The Western blot analysis results of total proteins of different engineered algae in the examples of the present invention are shown.

[0028] Figure 4 1 is the growth curve of each algae strain in the embodiment of the present invention.

[0029] Figure 5 These are the results of Western blot analysis of total proteins of different engineered algae of Chlamydomonas reinhardtii that were secondary-transformed into HLF in the examples of the present invention.

[0030] Figure 6In the embodiment of the present invention, different concentrations of Fe are added 2+ Western blot analysis results of total protein of engineered algae HLF6-18.

[0031] Figure 7 These are the Western blot analysis results of the total protein of the engineered alga HLF6-18 when different amino acids were added in the examples of the present invention.

[0032] Figure 8 The Western blot analysis results of total protein of the engineered algae HLF6-18 when different amounts of carbon sources were added in the examples of the present invention.

[0033] Fig. 9 The Western blot analysis results of total protein of the engineered alga HLF6-18 when different substrates were added in the examples of the present invention.

[0034] Fig.10 The Western blot analysis results of total protein of engineered algae HLF6-18 in different culture modes in the examples of the present invention are shown.

[0035] Fig.11 The results of Western blot analysis of the human lactoferrin LTF standard and the total protein of the engineered alga HLF6-18 in the examples of the present invention are shown. DETAILED DESCRIPTION

[0036] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. The specific implementation methods described herein are only used to explain the present invention, but not to limit the present invention.

[0037] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0038] Chlamydomonas reinhardtii, also known as green yeast, has passed the GRAS certification of the U.S. Food and Drug Administration and has been included in the Chinese food list. It is a safe-grade microalgae. It has a clear genetic background and is a new type of heterologous gene expression system. It has the two advantages of eukaryotic protein folding, post-translational modification and easy gene manipulation, which is conducive to the transformation and expression of exogenous genes. In addition, the use of algae for heterologous expression of target products has the advantages of short culture time, high cell density, and high nutritional value. It can also reduce the cost of extraction and purification and improve the competitive advantage of protein extraction from algae.

[0039] The present invention provides a method for constructing a microalgae engineering strain, comprising: transforming a target gene into Rhine microalgae at least twice to construct a microalgae engineering strain, and each time the target gene transformed into Rhine microalgae is successfully expressed, and the target gene is a gene capable of expressing human lactoferrin. Specifically comprising:

[0040] (1) Constructing a recombinant expression vector containing the human lactoferrin gene HLF;

[0041] (2) The recombinant expression vector containing HLF is transferred into Chlamydomonas reinhardtii by means of the glass bead method, and the HLF Chlamydomonas reinhardtii mutant is screened to obtain the engineered algae strain that synthesizes the recombinant human lactoferrin.

[0042] More specifically, the following steps are included:

[0043] Vector design and construction: The human lactoferrin HLF gene (GenBank: KT006756.1) and its encoded amino acid sequence (GenBank: ANC33015.1) were used to optimize introns and codons using BiBiServj (https: / / bibiserv.cebitec.uni-bielefeld.de / intronserter), and the optimized sequence was constructed into the Chlamydomonas reinhardtii expression vector. Preferably, the HLF expression vector uses PsaD as the promoter, 3HA is fused after the HLF sequence, and paromomycin is used as the resistance screening marker.

[0044] Genetic transformation: The vector is transferred into Chlamydomonas reinhardtii using the glass bead method, and plate resistance screening is performed. Preferably, Chlamydomonas reinhardtii is cultured on UVM4 for 2-3 days in the middle of the logarithmic phase (light green), and the HLF gene is transferred using the bead milling method.

[0045] Screening and identification: The algae strains grown in the resistance plate were verified by colony PCR using HLF as the target fragment, and the positive strains at the DNA level were detected by protein immunoblotting. Preferably, the primers are designed with the HLF nucleic acid sequence as the template; the WB test is to detect the HA screening tag, the primary antibody is Mouse Anti-HA Tag, and the secondary antibody is Goat Anti-Mouse IgG H&L / HRP.

[0046] Optimize and improve the yield of recombinant HLF: by re-transferring the HLF expression vector, the transcription level of the HLF gene is increased, thereby increasing the expression of the HLF protein; by optimizing the supply of iron ions to increase the expression of the HLF protein; by optimizing the supply of characteristic amino acids to increase the lactoferrin content; by optimizing the carbon source content to increase the lactoferrin content; by amplifying the 5L fermentation tank to increase the lactoferrin content. Preferably, the HLF expression vector in Chlamydomonas is re-transformed to replace the vector with the paromomycin resistance screening tag with hygromycin; more preferably, the optimized iron ion concentration is 0.3 mM; the characteristic amino acid is valine; and the optimized carbon source content is 6g / L.

[0047] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following embodiments are provided for further explanation:

[0048] The Chlamydomonas reinhardtii used for method verification in the specific embodiment of the present invention is Chlamydomonas reinhardtii cell wall-deficient algae UVM4, and HLF is a human lactoferrin gene optimized according to the codon preference of Chlamydomonas reinhardtii.

[0049] The formula of TAP medium: TAP medium includes 2.42 g Tris, 25 mL TAP-Salts (15 g NH4Cl, 4 g MgSO4·7H2O and 2 g CaCl2·2H2O dissolved in water and fixed to 1 L), 1 mL phosphate solution (288 g K2HPO4 and 144 g KH2PO4 dissolved in water and fixed to 1 L) and 1 mL Trace trace element mixture (50 g Na2EDTA·2H2O, 22 g ZnSO4·7H2O, 11.4 g H3BO3, 5 g MnCl2·4H2O, 5 g FeSO4·7H2O, 1.6 g CoCl2·6H2O, 1.6 g CuSO4·5H2O and 1.1 g (NH4)6Mo7O 24 ·4H2O dissolved in water, adjust pH to 6.5-6.8, and make up to 1 L) dissolved in 950 mL of water, adjust pH to about 7 with glacial acetic acid, and make up to 1 L.

[0050] Example 1

[0051] This example involves transformation and screening of human lactoferrin in Chlamydomonas reinhardtii.

[0052] 1. Construction and transformation of recombinant expression vector

[0053] (1) The expression vector used in this experiment is pHK263, which contains the PasD promoter sequence and the Paromycin resistance gene.

[0054] The human lactoferrin HLF gene (GenBank: KT006756.1) and its encoded amino acid sequence (GenBank: ANC33015.1), as shown in SEQ ID NO: 1, were optimized for introns and codons using the BiBiServj website (https: / / bibiserv.cebitec.uni-bielefeld.de / intronserter). The optimized HLF gene sequence is shown in SEQ ID NO: 2. The sequence was sent to the company to synthesize a DNA plasmid, namely pET28a-HLF.

[0055] SEQ ID NO: 1:

[0056] MKLVFLVLLFLGALGLCLAGRRRRSVQWCAVSQPEATKCFQWQRNMRKVRGPPVSCIKRDSPIQCIQAIAENRADAVTLDGGFIYEAGLAPYKLRPVAAEVYGTERQPRTHYYAVAVVKKGGSFQLNELQGLKSCHTGLRRTAGWNVPIGTLRPFLNWTGPPEPIEAAVARFFSASC VPGADKGQFPNLCRLCAGTGENKCAFSSQEPYFSYSGAFKCLRDGAGDVAFIRESTVFEDLSDEAERDEYELLCPDNTRKPVDKFKDCHLARVPSHAVVARSVNGKEDAIWNLLRQAQEKFGKDKSPKFQLFGSPSGQKDLLFKDSAIGFSRVPPRIDSGLYLGSGYFTAIQNLRKSE EEVAARRARVVWCAVGEQELRKCNQWSGLSEGSVTCSSASTTEDCIALVLKGEADAMSLDGGYVYTAGKCGLVPVLAENYKSQQSSDPDPNCVDRPVEGYLAVAVVRRSDTSLTWNSVKGKKSCHTAVDRTAGWNIPMGLLFNQTGSCKFDEYFSQSCAPGSDPRSNLCALCIGDEQG ENKCVPNSNERYYGYTGAFRCLAENAGDVAFVKDVTVLQNTDGNNNEAWAKDLKLADFALLCLDGKRKPVTEARSCHLAMAPNHAVVSRMDKVERLKQVLLHQQAKFGRNGSDCPDKFCLFQSETKNLLFNDNTECLARLHGKTTYEKYLGPQYVAGITNLKKCSTSPLLEACEFLRK

[0057] SEQ ID NO:2:

[0058]

[0059] (2) Using pET28a-HLF as a template and primers HLF-F1 and HLF-R1 as primers, the HLF gene sequence was amplified using the high-fidelity enzyme KOD. The PCR reaction system is shown in Table 1.

[0060] Table 1

[0061] Reagents Dosage HLF-F1 2 μL HLF-R1 2 μL Plasmids 1 μL KOD One PCR Master Mix 25 μL <![CDATA[ddH2O]]> Make up to 50 μL Total volume 50 μL

[0062] Table 2

[0063]

[0064] (3) Use N Ⅰ and EcoR The expression vector pHK263 was double-digested with restriction endonucleases Ⅰ, and the enzyme digestion system is shown in Table 3.

[0065] Table 3

[0066] Reagents Dosage Ⅰ 1 μL Ⅰ 1 μL Plasmids 1 μg 10×rCutSmart Buffer 5 μL <![CDATA[ddH2O]]> Make up to 50 μL Total volume 50 μL

[0067] The above reagents were added step by step into a 200 μL sterile PCR tube, mixed, and reacted at 37°C for 30 min.

[0068] After the reaction, the PCR products and enzyme digestion products were detected by 1% agarose gel electrophoresis (W / V). The PCR products with a single target band and correct size were recovered using New Gel Mini Purification Kit.

[0069] The recovered target gene fragments were homologously recombined with the expression vector fragments, and the system is shown in Table 4.

[0070] Table 4

[0071] Reagents Dosage HLF gene fragment 1.5 μL Expression vector pHK263 fragment 3.5 μL 2×HiFi Seamless Cloning Mix 5 μL Total volume 10 μL

[0072] The above reagents were added to a 200 μL sterile PCR tube in sequence, reacted at 50°C for 20 min, and then the homologous recombination products were transformed into competent cells.

[0073] The monoclonal colonies were picked up with a sterile pipette tip, and 2× SuperTaq PCR StarMix (Dye) was used for colony PCR identification and screening. The PCR products were detected by 1% agarose gel electrophoresis. The bacterial solution with the correct amplified fragment size was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The monoclonal colonies with the correct sequencing results were preserved separately. Thus, the human lactoferrin plasmid pHK263-HLF suitable for expression in Chlamydomonas reinhardtii cells was obtained. Subsequently, the plasmid pHK263-HLF was transformed into UVM4 engineered algae by the glass bead method, and screened on a TAP plate containing paromomycin until the transformants grew out.

[0074] Glass bead method transformation steps:

[0075] ① Linearize the plasmid, perform single enzyme digestion, and directly transform. The reaction system is shown in Table 5

[0076] Table 5

[0077] Reagents Dosage Ⅰ 1 μL Plasmids 500 ng 10×rCutSmart Buffer 2 μL <![CDATA[ddH2O]]> Make up to 20 μL Total volume 20 μL

[0078] ②Inoculate 100 μL of algae into 25 mL of TAP liquid medium at 25°C and 80 μmol / (m 2 ·s) light intensity for 3-4 days as seed solution. Centrifuge 1 mL, remove the supernatant, and keep 100 μL of algae solution to spread on TAP plate. 2 ·s) light intensity for 2-3 days (light green), and then proceed to the next transformation: add 130 μL of pre-cooled TAP (2% sucrose), 20 μL of enzyme-cut fragments, transfer to a 1.5 mL centrifuge tube, add 0.3 g of sterilized glass beads; transfer to a 15 mL centrifuge tube, contain 10 mL of TAP (2% sucrose) and culture overnight (dark treatment); remove the supernatant, add to 3 mL of TAP+0.5% agar+2% sucrose, mix well, spread on a paromomycin resistance plate, and culture upside down for one week until a single algae colony appears.

[0079] 2. DNA level identification of transgenic algae strains

[0080] A single algal colony was picked and inoculated into TAP liquid culture medium containing Par, and the genomic DNA of each single algal colony was extracted using StarSpinUniversal DNA Kit according to the instructions for PCR analysis.

[0081] Take 1 μL genomic DNA as template, and perform PCR verification using actin-F / actin-R, HLF-F2 / HLF-R2 and Par-F / Par-R as primers. Primers actin-F / actin-R are used as internal reference, and HLF-F2 / HLF-R2 and Par-F / Par-R are used to detect the presence of HLF and Par. Primer sequences and PCR reaction systems are shown in Tables 6 and 7.

[0082] Table 6

[0083]

[0084] Table 7

[0085] Reagents Dosage 2×SuperTaq PCR StarMix(Dye) 10 μL actin-F / HLF-F2 / Par-F 1 μL actin-R / HLF-R2 / Par-R 1 μL DNA template 1 μL <![CDATA[ddH2O]]> Make up to 20 μL Total volume 20 μL

[0086] PCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s; annealing at 58°C for 15 s; extension at 72°C for 1 min; 33 cycles; extension at 72°C for 5 min.

[0087] After the PCR reaction, 1% agarose gel electrophoresis was used to detect the transgenic algae transferred into HLF. The PCR identification results were as follows: Figure 1 As shown, M represents marker protein, U4 represents wild bacteria, and water represents negative control. Among them, 4 engineered algae can amplify bands with the same size as the positive control; these results show that the HLF gene exists in the above-mentioned engineered algae.

[0088] 3. Identification of transcriptional levels of transgenic algae strains

[0089] (1) Use Eastep® Super Total RNA Extraction Kit to extract total RNA from algae strains;

[0090] Prepare the DNase I incubation solution according to Table 8. The following is the amount required to extract one tube of RNA.

[0091] Table 8

[0092] Reagents volume 10× DNase I incubation solution 5 μL DNase I 5 μL Nuclease-free water 40 μL

[0093] ① Add 50 μL of DNase I incubation solution to the center of the adsorption membrane and leave it at room temperature for 15 min.

[0094] ② Add 600 μL RNA washing solution, centrifuge at 12000 rpm for 45 s, and discard the filtrate.

[0095] ③ Add 600 μL RNA washing solution, centrifuge at 12,000 rpm for 45 s, discard the filtrate, reinstall the centrifuge column on the collection tube, and centrifuge at 12,000 rpm for 2 min.

[0096] ④ Transfer the centrifuge column to the elution tube, add 50 μL of nuclease-free water to the center of the centrifuge column membrane, let it stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 1 minute, determine the concentration using NanoDrop, and store at -80°C.

[0097] (2) Synthesis of algal cDNA

[0098] HiScript® Ⅲ RT SuperMix for qPCR (+gRNA wiper) was used for reverse transcription reaction. 1 μg of total RNA was pipetted into an RNase-free centrifuge tube, and reaction reagents were added in sequence to remove genomic DNA (gDNA). The reaction system is shown in Table 9.

[0099] Table 9

[0100] Reagents Dosage <![CDATA[RNase-free ddH2O]]> to 16 μL 4×gDNA wiper Mix 4 μL Template RNA 1 μg

[0101] Use a pipette to gently mix, incubate at 42°C for 2 min, then add reverse transcription reagents to synthesize cDNA. The reaction system is shown in Table 10.

[0102] Table 10

[0103] Reagents Dosage 5×HiScript Ⅲ qRT SuperMix 4 μL Reaction solution of step 1 16 μL Total volume 20 μL

[0104] Mix by gently blowing with a pipette, incubate at 37℃ for 15 min, incubate at 85℃ for 5 s, terminate the reaction at 4℃ to complete the cDNA synthesis, and store the synthesized cDNA in a -20℃ refrigerator

[0105] (3) qRT-PCR reaction of algae strains

[0106] The expression level of the target gene was detected using the SYBR Green Ⅰ fluorescent dye method, with actin as an internal reference. The reaction system is shown in Table 11, and the primers are shown in Table 12.

[0107] Table 11

[0108] Reagents Dosage 2×ChamQ Universal SYBR qPCR Master Mix 10 μL Primer 1 (10 μΜ) 0.4 μL Primer 2 (10 μΜ) 0.4 μL Template cDNA 0.5 μL <![CDATA[ddH2O]]> to 20 μL Total volume 20 μL

[0109] Table 12

[0110]

[0111] qRT-PCR reaction conditions: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 10 s; 60℃ annealing and amplification for 30 s; 40 cycles. Melting curve setting: 95℃ 15 s; 60℃ 60 s; 95℃ 15 s.

[0112] The above qRT-PCR experiment was used to detect the mRNA expression of human lactoferrin gene in different transgenic algae. The results are as follows Figure 2 As shown, strain HLF6 showed the highest expression level.

[0113] 4. Protein level identification of transgenic algae strains

[0114] (1) Extraction of total protein from algae

[0115] ① On a clean bench, take 1 mL of seed solution and inoculate it into 150 mL of fresh TAP liquid medium and culture it until the late logarithmic growth stage;

[0116] ② Collect 25 mL of algae liquid and centrifuge at 6000 rpm for 5 min. Add 2 mL of protein lysis buffer (20 μL of PMSF has been added in advance) and incubate at 4°C for 15 min. Shake occasionally during incubation to fully lyse the algae cells.

[0117] ③ Centrifuge at 12,000 rpm for 10 min at 4°C and collect the supernatant as the total protein sample.

[0118] (2) Western blot experiment of algae protein

[0119] The protein gel was prepared using PAGE Gel Fast Preparation Kit from YAZYME, and then the gel was used for Western blot experiments. The target band was detected using Immobilon® Western Chemiluminescent HRP Substrate. Mix the A and B solutions of the ECL reagent in advance according to the instructions. Pipette 1 mL of ECL reagent onto the membrane, incubate for 1 min, then expose using a developer, take pictures, and save the results for analysis.

[0120] Western blot analysis results of HLF engineered algae are shown in Figure 3 As shown in the figure, specific bands were detected in the total protein samples of engineered algae 6, 9, 24, and 40, and their sizes (~80 kDa) were consistent with expectations, while no specific bands were found in the negative control. The results showed that the proteins were expressed normally in each transformant.

[0121] 5. Physiological characteristics of transgenic algae strains

[0122] Wild algae (UVM4) and HLF engineered algae were inoculated into liquid TAP medium and placed in a continuous light incubator (25°C, 80 μmol / (m 2 ·s) for one week, during which algae liquid was sampled regularly and the OD value was measured using a UV spectrophotometer. A growth curve was drawn based on the daily OD results. Figure 4 As shown, with the increase of culture time, HLF engineered algae showed a growth rate similar to that of wild algae. This result indicated that HLF engineered algae had no effect on the growth of algae strains.

[0123] 6. Yield analysis of transgenic algae strains

[0124] On the clean bench, aspirate TAP liquid medium and dispense it into a 24-well plate. Use a sterile pipette tip to pick up the transformed algae and drop them into the medium. Place it at 25°C and add 80 μmol / (m 2 ·s) light intensity to the logarithmic growth phase as seed solution. The seed solution was inoculated into fresh TAP liquid medium and cultured to the stable phase. Western blot experiments were performed on it (refer to the above total protein extraction and Western blot experimental methods), and the gray value results were evaluated.

[0125] Example 2

[0126] This example optimizes the culture conditions of transgenic Chlamydomonas.

[0127] 1. Replace the paromomycin resistance screening tag with hygromycin to construct a new human lactoferrin expression vector pHK263-HLF-HPT, and transfer it into the algae strain HLF6 again and identify it (refer to the construction and identification method in Example 1, where the primer sequences and PCR reaction system for DNA level identification are shown in Tables 13 and 14.)

[0128] Genomic DNA was used as a template, and PCR verification was performed using actin-F / actin-R, HLF-F2 / HLF-R2, and HPT-F / HPT-R as primers. Primers actin-F / actin-R were used as an internal reference, and HLF-F2 / HLF-R2 and HPT-F / HPT-R were used to detect the presence of HLF and HPT.

[0129] The results of gene and protein identification were as follows Figure 5 As shown, the algal strain HLF6-18 with higher expression of human lactoferrin was obtained.

[0130] Table 13

[0131]

[0132] Table 14

[0133] Reagents Dosage 2×SuperTaq PCR StarMix(Dye) 10 μL actin-F / HLF-F2 / HPT-F 1 μL actin-R / HLF-R2 / HPT-R 1 μL DNA template 1 μL <![CDATA[ddH2O]]> Make up to 20 μL Total volume 20 μL

[0134] 2. Fe 2+ Determination of the production of human lactoferrin engineered strains under concentration changes: The human lactoferrin engineered strain HLF6-18 was inoculated with different concentrations of 0.02 mM, 0.04 mM, 0.06 mM, 0.08 mM, 0.1 mM, 0.2 mM, 0.3 mM, and 0.4 mM Fe 2+ 150 mL of sterile liquid TAP medium was placed at 25°C and heated to 80 μmol / (m 2 ·s) light intensity for 4 days. Figure 6 As shown, the engineered algae strain was added with 0.3 mM Fe 2+ The human lactoferrin obtained in the culture medium is about 2.5 times that in TAP medium. 2+ Cultivation in the culture medium significantly increased the production of human lactoferrin in the engineered algal strain.

[0135] 3. Determination of the yield of human lactoferrin engineered strains under amino acid induction: The human lactoferrin engineered strain HLF6-18 was inoculated into 150 mL sterile liquid TAP medium with different amino acids added at a total concentration of 8 mM: leucine Leu, valine Val, leucine (4 mM) + valine 4 mM (L+V), and placed at 25°C at 80 μmol / (m 2 ·s) light intensity for 4 days. Figure 7 As shown, the human lactoferrin obtained by the engineered algae strain in the medium supplemented with valine (Val) is about 1.5 times that obtained in the TAP medium, indicating that the culture medium supplemented with valine (Val) can significantly increase the production of human lactoferrin in the engineered algae strain.

[0136] 4. Determination of the yield of human lactoferrin engineered strains under varying carbon source content: The human lactoferrin engineered strain HLF6-18 was inoculated into 150 mL of sterile liquid TAP medium supplemented with different contents of 2 g / L, 4 g / L, 6 g / L, 8 g / L, and 10 g / L of sodium acetate as a carbon source, and placed at 25°C and 80 μmol / (m 2 ·s) light intensity for 4 days. Figure 8 As shown, the engineered algae strain accumulated the most human lactoferrin in the culture medium supplemented with 6 g / L sodium acetate, which was about three times that in TAP medium.

[0137] 5. Determination of the yield of human lactoferrin engineered strains under optimized culture conditions: The engineered strain HLF6-18 was inoculated into culture medium supplemented with different substrates: 8 mM valine Val, 0.3 mM Fe2+ , 6 g / L sodium acetate and 8 mM valine Val, 0.3 mM Fe 2+ , 6 g / L sodium acetate in a 150 mL sterile liquid TAP medium at 25 °C, 80 μmol / (m 2 ·s) light intensity for 4 days. Fig. 9 Under the comprehensive culture conditions, the maximum content of human lactoferrin was 3.5 times that before the conditions were optimized.

[0138] 6. Determination of the yield of human lactoferrin engineered strain under fermentation amplification culture conditions: The engineered strain HLF6-18 was inoculated into a culture medium supplemented with 8 mM valine Val, 0.3 mM Fe 2+ , 6 g / L sodium acetate in 5 L of liquid TAP medium at 25°C, 80 μmol / (m 2 ·s) light intensity for 4 days. Fig.10 Under the optimal culture conditions, the maximum content of human lactoferrin was 7 times that before the conditions were optimized.

[0139] 7. Quantitative determination of the yield of human lactoferrin engineered strains: Human lactoferrin standard (LTF, 90801ES03) was used to extract protein samples from LTF and unoptimized HLF6-18 algae cells at concentrations of 1, 1.5, 2, 3, 3.5, 4, and 4.5 mg / L, respectively, and protein electrophoresis was performed, and protein immunoblotting analysis was performed using the HLF primary antibody (bsm-33157M) (refer to the Western blot experimental method of algae protein in Example 1). The HLF concentration standard curve was constructed using the gray value and the known loading concentration, as shown in FIG. Fig.11 According to the standard curve calculation, the HLF protein content reached 0.3% of the cell dry weight.

[0140] In summary, the present invention transforms the human lactoferrin HLF gene into Chlamydomonas reinhardtii UVM4, introduces the human lactoferrin synthesis pathway, and after DNA level screening, the expression level of the transgenic transformant is analyzed by Western Blot, and the specific band sizes of 80 kDa are obtained, which are consistent with the size of the target protein. High-expression HLF engineered algae 6, 9, 24 and 40 are obtained. The culture conditions for obtaining the highest yield of HLF engineered algae strains were explored: a new expression vector was constructed and transferred into the engineered algae HLF6 to obtain the engineered algae strain HLF6-18 with a higher expression level. On this basis, the engineered algae strain HLF6-18 was inoculated into the TAP medium and 0.3 mMFe was added. 2+ , valine and 6 g / L sodium acetate (carbon source), at 25 °C, 80 μmol / (m 2·s) light intensity, the human lactoferrin production was increased by about 3.5 times compared with that before the optimization of culture conditions, and then 5 L fermentation was carried out to expand the culture, and finally the maximum production of human lactoferrin was increased by about 7 times compared with that before the optimization of culture conditions, and the content reached 1.1% of the cell dry weight.

[0141] The number of modules and processing scales described here are used to simplify the description of the present invention. Applications, modifications and variations of the present invention are obvious to those skilled in the art.

[0142] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A method for efficiently synthesizing recombinant human lactoferrin, characterized in that: The steps include: 1) transforming a target gene into Rhineland-Palatinate microalgae at least twice to construct an engineered microalgae bacterium, and the target gene transformed into Rhineland-Palatinate microalgae at least twice is successfully expressed, wherein the target gene is a gene capable of expressing human lactoferrin, and its nucleotide sequence is the gene sequence shown in SEQ ID NO: 2; 2) The microalgae engineering bacteria are inoculated into a liquid TAP medium and cultured under light at a temperature of 20-30°C, and the algae liquid is harvested to obtain recombinant human lactoferrin from the algae liquid. The liquid TAP medium is additionally supplemented with Fe 2 + , valine and 2 ~10 g / L sodium acetate.

2. The method for efficiently synthesizing recombinant human lactoferrin according to claim 1, characterized in that: The TAP liquid medium was additionally supplemented with 0.3 mM Fe 2+ , 8 mM valine and 6 g / L sodium acetate.

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