WO2013166863A1

WO2013166863A1 - Preparation method of competent host cells and uses thereof

Info

Publication number: WO2013166863A1
Application number: PCT/CN2013/000567
Authority: WO
Inventors: 张成岗; 范磊; 吴永红; 李�雨; 周浪; 高艳; 李伟光; 李志慧
Original assignee: 苏州科景生物医药科技有限公司; 北京百乐宁生物技术有限公司; 中国人民解放军军事医学科学院放射与辐射医学研究所
Priority date: 2012-05-11
Filing date: 2013-05-13
Publication date: 2013-11-14
Also published as: CN103387946A

Abstract

Disclosed in the present invention are a preparation method of competent host cells and a method for rapidly and efficiently transferring an exogenous substance into the host cells, and uses thereof, wherein the core of the method lies in that: the competent host cells are obtained by treating the host cells using conventional vacuum freeze drying technology; then the competent host cells are rehydrated by an aqueous solution containing the exogenous substance; and microorganisms transformed with the exogenous substance are obtained, by cultivating the rehydrated host cells placed in a constant temperature incubator and transferring the host cells into a conditioned medium, and then screening same. The exogenous substance can be transferred into the host cells rapidly by means of the present invention, which has the following advantages: easy to operate, with little injury, with a low cost, etc. The competent cells in batches can be obtained very easily via the present invention, which is suitable for the application fields of research into the activity and functions of exogenous substances and the like.

Description

Method for preparing competent host cells and application thereof

The invention belongs to the field of biological products and biotechnology applications, and in particular to a method for transferring foreign substances into host cells.

Background technique

In the field of biotechnology, lateral gene transfer refers to the process by which organisms transfer genetic material levels to other cells rather than to their offspring. It is an important means of studying the function of foreign genes, especially at the microbial level (such as viruses). , bacteria, fungi, plant cells, animal cells, etc.) are very common. A bacterium often acquires new traits by obtaining some genetic material from other bacteria. For example, the current use of antibiotics in large quantities leads to the genes encoding decomposable antibiotics in different bacteria. Spread laterally to form resistant bacteria. In the laboratory, the nature of this genetic material to spread and spread between different individuals is often used to study the function of exogenous DNA and other biologically active substances. However, cells including bacteria, fungi (such as yeast), plant cells, animal cells, etc. usually have cell membranes that restrict the entry of foreign substances, so how to change these cells to acceptable by a certain technical treatment. The state of exogenous substances, ie, the competent state, is an important research content in the fields of bioengineering and biotechnology.

At present, the commonly used methods for promoting the transformation of foreign substances into host cells are: using divalent metal ions

(such as calcium ion) Under certain temperature conditions (such as 42'C), the host cell will become more porous due to thermal stimulation, so that it is easy to ingest foreign substances (such as DNA) (Mei Yunjun, Chen Xiangdong) , Xie Zhixiong, etc. Ca~

(2 + ) Effects on the induction of exogenous DNA by E. coli. Journal of Wuhan University: Science Edition, 2012, 58 (4): 354-358. ), or using a high-voltage electric field to induce transient perforation of host cell membranes (Zhang Yang, Wang Zhiqiang, Liu Bin et al. Exploratory conditions for efficient electroporation of DH10B strain. Chinese Journal of Biotechnology, 2007, 23 (2): 347-351. However, these methods have the disadvantages of complicated operation, large damage to cells, high operating cost, and low conversion efficiency. How to establish a rapid and efficient method for transforming (transferring) foreign substances into host cells is one of the key issues in the field of bioengineering and biotechnology.

Summary of the invention

Based on the above problems, an object of the present invention is to provide a method for preparing a rapid and efficient competent host cell, and a method for transferring a foreign substance into a host cell.

The preparation method of the competent host cell provided by the invention comprises the following steps -

1) purifying, resuscitating and pretreating host cells;

2) The host cell is treated by a vacuum freeze-drying technique to obtain a competent host cell.

The method for transferring a foreign substance into a host cell provided by the present invention further comprises the following steps: 3) preparing an aqueous solution of the exogenous substance to be transferred;

4) The competent host cells obtained in the step 2) are rapidly rehydrated to transfer the foreign substances into the host cells, and the host cells transformed with the foreign substances are screened by the conditioned medium to obtain the foreign substance-transformed bacteria.

In the above method, the host cell in the step 1) may be any prokaryotic or eukaryotic host cell, such as a bacterial cell, a fungal cell, a plant cell, and an animal cell. Since bacteria (most commonly E. coli) and yeast have the advantages of clear genetic background, high target gene expression level, mature expression system, easy cultivation, low cost, and the like, Escherichia coli and yeast are used in the examples of the present invention.

The purified host cell means that the original host cell is streaked on the surface of the solid medium to obtain a monoclonal; the resuscitating the host cell means that the purified host cell is re-cultured for a certain period of time to re-enter the state from the quiescent state to the growth state; Pre-treating a host cell means expanding the resuscitated host cell to a corresponding concentration. Specifically, the steps 1) of purifying, resuscitating, and pretreating host cells are as follows -

(1) Purification of host cells: The original host cells are picked with an inoculating loop, and the partitions are streaked into a solid medium (LB solid medium (suitable for E. coli) formulation suitable for culturing host cells: tryptone 10 g/L, yeast extract 5 _g / L, NaCl 10g / L, agar powder 15g / L; YPD solid medium (suitable for yeast) Formula: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agar powder 20g / L), in Under suitable conditions (E. coli 37'C, 12~16h; yeast culture 30'C, 48h), the host cell monoclonal is obtained;

(2) Resuscitation of host cells: Pick up host cells and inoculate them into 5 mL of liquid medium suitable for host cells (LB medium (suitable for E. coli) formula: trypsin 10 _g / L, yeast extract 5 g / L, NaCl 10 g /L _; YPD medium (suitable for yeast) formula: yeast extract 10g / L, peptone 20g / L, glucose 20g / L), at a suitable temperature (E. coli 37'C _; yeast culture 30'C) 220rpm resuscitation overnight (12~16 hours);

(3) Pretreatment of host cells: The resuscitated host cells are inoculated to 100 mL of fresh liquid medium (LB medium (suitable for E. coli) formulation at a ratio of 1:100 by volume: trypsin 10 g/L, yeast extract 5 g /L, NaCl 10g / L; YPD medium (suitable for yeast) formula: yeast extract 10g / L, peptone 20g / L, glucose 20g / L), at the appropriate temperature (E. coli 37 'C _; yeast culture 30 Ό) Shake the bacteria to 0D at 220 rpm, 0. 6~1. 0; Pipette 500 μl of the bacterial solution into 1. 5 mL EP tube, _80'C refrigerator for 12 to 24 hours or more.

In the above method, the method of vacuum freezing the dried host cells in the step 2) is: using any vacuum freeze dryer on the market or a self-processing vacuum freeze dryer to use the host cells treated in the step 1) The vacuum freeze dryer was pre-cooled for 30 to 120 minutes, and after the temperature reached -54 ° C and stabilized at this temperature, the host cells were vacuum-dried for 12 to 24 hours or more.

In the above method, the exogenous substance used in the step 3) includes a biologically active substance such as a nucleic acid (deoxyribonucleic acid (DNA), ribonucleic acid (NA)), a protein, an oligopeptide, an amino acid or the like. The aqueous solution of the exogenous substance used ddH ₂ 0. The amount of the exogenous substance may vary depending on the specific research purpose (for example, the amount of the plasmid DNA is not less than O. Olng: for other exogenous substances to be transformed, those skilled in the art may determine the exogenous substance according to the published literature. The amount of the prepared aqueous solution of the exogenous substance is kept at 37 ° C for 30 to 60 minutes.

In the above method, the method for rapidly rehydrating the competent host cells in the step 4) is: rapidly adding the competent host cells of the step 2) to the step 3) preparing and heat-treating the aqueous solution containing the foreign substances, Gently mix and incubate at 37 ° C for 30 minutes, place at a suitable temperature (37 ° E. coli; yeast culture 30 ° C) in an incubator at 220 rpm for 45 to 60 minutes; use of aqueous solution containing exogenous substances and freeze The volume of the host cells before drying is equal.

The conditioned medium for screening in step 4) is: LB solid medium (suitable for E. coli), and the formula is: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar powder 15g/L, final concentration 100-200 μg/mL antibiotic for foreign substances: YPD solid medium (suitable for yeast), formula: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agar powder 20g / L, An antibiotic suitable for exogenous substances at a final concentration of 100-200 μg/mL. The choice of antibiotics is related to the type of exogenous substance, and can be determined by those skilled in the art based on the exogenous substance reference to the published literature.

The process of screening competent host cells in step 4) is: inoculating or coating the reconstituted competent host cell broth into the conditioned medium at a suitable temperature (E. coli 37'C; yeast culture 30'C) After a constant temperature culture for 12 to 16 hours, a monoclonal antibody is obtained, that is, a host cell transformed with a foreign substance, that is, a foreign substance-converting bacteria is obtained.

Competent host cells and exogenous substance-transformed bacteria prepared in the above methods are also included in the present invention. Moreover, the competent host cells can maintain biological activity at the temperature of O-4'C, which is convenient for storage and transportation, and currently the competent cells in the field can only be transported under the condition of a thousand ice (-70'C).

The invention utilizes the vacuum freeze-drying technology to prepare the competent host cells, and can rapidly and efficiently convert (transfer) the foreign substances to the competent host cells, and the core thereof is the vacuum freeze-drying technology and the rehydration technology. The invention first freeze-drying the host cell under vacuum to obtain a competent host cell, and then adding the aqueous solution of the exogenous substance to be transformed to the competent host cell for rehydration, and placing the host cell after rehydration at a constant temperature. After culturing in the incubator, the cells are transferred to a conditioned medium containing resistance pressure for screening, and the biochemical indicators can be used to confirm whether the transformation is successful. The invention can not only realize rapid transformation of the host nucleic acid into the host cell, but also realize the rapid transformation of the host cell by the biological active substances such as exogenous proteins, amino acids, sugars and the like, and the method has the advantages of simple operation, small damage and low cost. Etc. Through the practice of the present invention, not only a batch of competent host cells (ie, freeze-dried host cells, in a state in which foreign DNA and other foreign substances are easily accepted) can be obtained simply and quickly, and can be used for subsequent biological functions. The research can also provide important technical research means for transmembrane transport of exogenous substances, and then provide key technologies for functional research of exogenous substances, which can significantly save research costs, so the invention has a wide range of applications before.

The present invention will be further described in detail below in conjunction with specific embodiments.

DRAWINGS

1 is a flow chart of a method for rapidly and efficiently transforming a foreign substance into a host cell by using a vacuum freeze-drying technique of the present invention.

Figure 2A shows the results of rehydration transformation of the prokaryotic expression vector pET28b-Tat at 37 °C.

Figure 2B shows the results of rehydration transformation of prokaryotic expression vector _P ET28b-Tat at 42 °C

Figure 3 shows the results of bacterial growth curve analysis of pET28b-Tat-EGFP after rehydration at 37 °C. Figure 4A shows the results of _S ET28b-Tat-EGFP induced expression by SDS-PAGE and immunoblotting. Figure 4B shows pET28b- Tat-EGFP-induced expression was detected by 6-hour SDS-PAGE and immunoblotting. Figure 5 shows the results of recombinant PH19 α recombination of plasmid _P UC19 at 37 °C. Figure 6 shows the plasmid pUC19 at 37 °C. Water-transformed Pichia pastoris GS115 competent results

detailed description

In view of the existing methods of lateral transfer of genes such as calcium ions and high voltage, which have the disadvantages of complicated operation, large damage to cells, high operation cost, and low conversion efficiency, the present invention strives to obtain a fast and efficient external source. A method of transforming a substance into a host cell.

The present invention utilizes vacuum freeze drying techniques to effect the conversion of exogenous substances into host cells.

The vacuum freeze-drying technology utilizes the principle of sublimation to realize the direct sublimation of solid water in biological tissues into a gas evaporation process under vacuum and low temperature conditions, which not only maintains the integrity of biological samples, but also maintains the activity of biological samples (Kang MS, Jang H, Kim MC, Kim MJ, Joh SJ, Kwon JH, Kwon YK: Development of a stabilizer for lyophilization of an attenuated duck viral hepatitis vaccine. Poult Sci 2010, 89(6) : 1167-1170; Chen C, Han D , Cai C, Tang X: An overview of liposome lyophilization and its future potential. Control Release 2010, 142 (3) : 299- 311. ). At present, freeze-drying technology has been widely used in the drying and long-term preservation of food, biological cells, tissues and drugs in the food industry, biology and pharmacy (Liu JH, Zhou J, Wang DM, Ouyang XL, Xing). YC, Lu FQ: [Experimental study on lyophilization of platelets]. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2007, 15 (5) : 1098-1101; Matejtschuk P: Lyophilization of proteins. Methods Mol Biol 2007, 368:59-72 Taniwaki L, Mendonca R, Cunha-Junior AS, Faraco Ah, Ribeiro JA, Scott IU, Jorge R: Effect of Lyophilization on the in vitro biological activity of bevacizumab. Eye (Lond) 2010). Using freeze-drying technology, not only freeze-drying and rehydration of prokaryotic cells can be achieved (Renoux G: [Lyophilization of Brucella strains.]. Ann Inst Pasteur (Paris) 1962, 103: 290-292), and eukaryotes can be realized. Lyophilization and rehydration of cells, such as yeast cells and mouse sperm cells (Guibert L, Brechot P: [Lyophilization of yeasts; first part.]. Ann Inst Pasteur (Paris) 1955, 88(6): 750- 768; Atkin L, Moses W, Gray PP: The preservation of yeast cultures by lyophilization. J Bacteriol 1949, 57(6) :575-578; Leirner AA, Tattini V, Jr. , Pitombo RN: Prospects in lyophilization of bovine pericardium Artif Organs 2009, 33(3):221-229) ₀ Researchers have found that freeze-drying technology can achieve long-term storage at room temperature in mouse organs, where nucleic acids and proteins do not degrade with storage time, tissue freezing It becomes bulky after drying and maintains structural integrity after rehydration (Yonghong Wu, Min Wu, Yanchun Zhang, Weiguang Li, Yan Gao, Zhihui Li, Zhaoyan Wang, Gert Lubec, Chenggang Zha Ng. Lyophilization is suitable for storage and shipment of fresh tissue samples without altering RNA and protein levels stored at room temperature. Amino Acids. 2012, 43(3): 1383-1388).

Vacuum freeze-drying technology not only maintains the integrity and activity of biological samples, but the inventors also found in the study: After lyophilization of bacterial cells (such as E. coli), a certain number of holes will appear in the cell membrane. At this time, if an aqueous solution of a foreign substance to be transformed (for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), protein, biologically active substance, and other substances) is added to the bacterial cells after vacuum freeze-drying treatment, The source material can quickly enter the bacterial cells along with the water, thereby achieving the purpose of transferring foreign substances into the cells. On this basis, the inventors proposed the following mechanism: Freeze-drying technology can be used to freeze-dry and rejuvenate E. coli cells; E. coli after lyophilization becomes bulky, the membrane pores are loose, and the membrane pores are quickly rehydrated. Closing; lyophilized E. coli can rapidly transform foreign substances into host cells during the rehydration process as the membrane pores are closed, and then the host bacteria transformed with the foreign substance can be obtained by screening the conditioned medium.

The following is further illustrated by the examples.

The methods used in the following examples are conventional methods unless otherwise specified, and specific procedures can be found in - "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3 ^rd Edition, 2001, NY, Cold Spring Harbor ).

The various biomaterials described in the examples are obtained by merely providing an experimental acquisition route. For the purpose of specific disclosure, it should not be a limitation on the source of the biological material of the present invention. In fact, the sources of biological materials used in the present invention are extensive, and any biological material that can be obtained without violating laws and ethics can be replaced by the instructions in the examples.

The embodiments are implemented on the premise of the technical solution of the present invention, and detailed embodiments and specific operation procedures are given. The embodiments will be helpful for understanding the present invention, but the scope of protection of the present invention is not limited to the following implementations. example. Indicator detection,

In the present example, the exogenous substance is exemplified by the plasmid pET28b-Tat-EGFP, and the host cell is exemplified by Escherichia coli BL21 (DE3). Referring to Figure 1, the following operations are performed to transfer foreign substances into host cells:

1) Purification, resuscitation and pretreatment of E. coli host cells

(1) Purification of host cells: Purification of E. coli host cell BL21 by LB solid medium (formulation: tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar powder 15 g/L) at 37 ° C under streak culture (DE3), obtaining a monoclonal cell of an E. coli host cell;

(2) Resuscitation of host cells: Pick up E. coli host cells and inoculate them into 5 mL of LB liquid medium (formulation: tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L), 37. C, 220 rpm resuscitation overnight (12 16 hours);

(3) Pretreatment of host cells: The resuscitated E. coli host cells are inoculated to 100 mL of fresh LB liquid medium (the same formula as in step (2)) at a volume ratio of 1:100, shaken at 37 ° C, 220 rpm. Bacteria 2 to 3 hours to 0D ₆ . . =0. 6~1. 0, Pipette 50CmL of the bacterial liquid into 1. 5mL EP tube, -80 Ό refrigerator pre-freezing for 12 to 24 hours or more.

2) Vacuum freeze-drying E. coli host cells

After pre-cooling the vacuum freeze-drying instrument (purchased from Beijing Songyuan Huaxing Technology Development Co., Ltd.) to a temperature of -54 °C and stabilizing, the E. coli host cells pretreated in step 1) were taken out from the -80 Γ low temperature refrigerator and quickly Place in a vacuum freeze dryer, close the inlet valve and open the vacuum pump after setting the lid. Set the temperature to -54 ° low temperature conditions, vacuum freeze the host cells for 12 to 24 hours or more, turn off the vacuum pump after drying, and take out the freeze-drying technique. Escherichia coli, the E. coli prepared by this method is "competent E. coli BL2KDE3"" (with the ability to accept the conversion of foreign substances), can be used immediately or -20 Ό for storage, or can also be 0-4 ° C Save and transport under conditions.

The following test results show that "competent E. coli BL21 (DE3)" has the ability to accept the transfer of foreign substances. When transforming a foreign substance into a host cell, continue with the following steps:

3) Preparation of an aqueous solution for the conversion of exogenous substances Firstly, the pre-transformed plasmid pET28b-TatTMEGFP was determined by ultraviolet spectrophotometer. (Construction method: 1. The cDNA fragment of EGFP was amplified by conventional PCR method; 2. The restriction endonuclease was digested with the target gene EGFP and the vector pET28b- Tat, agarose gel electrophoresis was recovered and ligated using T4 DNA ligase. References (Wu Y, Ren C, Gao Y, Hou B, Chen T, Zhang C. A novel method for promoting heterologous protein expression in Escherichia coli by fusion Amino Acids. 2010 Aug : 39 (3) : 811-20) or Chinese Patent Document 201010119397. 8 Method for promoting soluble expression of foreign proteins by using TAT core peptide and its specific expression vector Concentration of the construction process, and dilute it to lng/μ 用 with dd 0 , and add 5 μ 1 , 10 1 , 25 μ 1 , 50 μ 1 , 100 μ 1 plasmid pET28b-Tat-EGFP to 500 μ Mix 1 ddH ₂ 0 to prepare different concentrations of aqueous solution, and incubate in a constant temperature water bath at 37 ° C for 30 to 60 minutes.

4) The vacuum freeze-dried E. coli host cell ("competent E. coli BL2 DE3)" obtained in the above step 2) was rapidly rehydrated, and conditioned medium was used to obtain Escherichia coli transformed with foreign substances. Host cell:

(1) Rapid rehydration: The "competent E. coli BL21 (DE3)" obtained in the step 2) is rapidly added to the aqueous solution containing the plasmid pET28b-Tat_EGFP prepared by the step 3) of the incubation at 37 ° C, and the aqueous solution is freeze-dried. The former host cell is equal volume (500 μ 1 ), gently mixed and incubated at 37 ° C (or 42 ° C) for 30 minutes, placed in a 37 ° C incubator at 220 rpm shaking culture for 45 to 60 minutes;

(2) Screening: Take 100 μl of E. coli host cell solution after lyophilization and rehydration (in step 3) with different plasmid concentrations (in step 3), respectively, to the conditioned medium containing kanamycin. LB solid medium (formulation: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar powder 15g / L, final concentration 200 μg / mL antibiotic kanamycin), at 37' Incubate at a constant temperature for 12 to 16 hours, and select the grown monoclonal to obtain an E. coli host cell into which a foreign substance has been transformed, that is, "Tat-EGFP transforming bacteria".

During the experiment, the ability of the competent Escherichia coli BL2KDE3) to be transformed by the exogenous substance _P ET28b-Tat-EGFP was observed by observing the number of monoclonal antibodies. It was found that the plasmid pET28b Tat- was maintained at different temperatures of 37 ° C and 42 ° C. The amount of EGFP transfected with competent E. coli BL21 (DE3) increased with increasing plasmid concentration, but there was no significant difference between the two, see Figure 2A (under 37'C) and Figure 2B (at 42 °C) As shown, the subsequent rehydration conversion of the exogenous substance was carried out directly at 37 °C.

Verification of Tat-EGFP transformants continues the following:

5) Biochemical indicator detection of Tat-EGFP transformed bacteria in E. coli host cells transformed with foreign substances

(1) Dynamic monitoring of growth curve of Tat-EGFP transforming bacteria The above 300 μl "Tat-EGFP transformant" was diluted with LB liquid medium containing kanamycin (Kana ⁺ , final concentration 200 μg/mL) at a volume ratio of 1:10 and inoculated into the microbial growth curve. Microplates (350 μl/well) were monitored for real-time dynamic monitoring of microbial growth.

The monitoring results are shown in Figure 3. The first column in Figure 3 is a normal strain, which was not transformed with the pET28b-Tat-EGFP plasmid, so no clones were grown in the conditioned medium screen; the second to fourth columns were three sets of parallel replicates, all The "Tat-EGFP transforming bacteria" containing the plasmid of pET28b-Tat-EGFP all grew a single clone, indicating that the competent cell preparation method of the present invention was successful. It can be seen that Escherichia coli ("Tat EGFP transforming bacteria") which has been transformed with foreign substances can grow in Kana-resistant medium, and its growth curve is good; observation of colonies growing on the surface of solid medium indicates that it has been transformed The morphology of E. coli colonies with foreign substances is normal. Therefore, it was shown that the "competent Escherichia coli BL21 (DE3)" obtained in the step 2) (the Escherichia coli host cell obtained by vacuum freeze-drying) has no genetic alteration and can be used as a host cell for exogenous substance transformation.

(2) Detection of exogenous protein expression in Escherichia coli transformed with foreign substances

To further confirm whether the foreign protein in E. coli transformed with foreign substances can be expressed as expected, this step is to inoculate "Tat-EGFP transforming bacteria" into 5raL containing Kana (final concentration 200 ₈ /11^). 1^Liquid medium and resuscitation overnight (12~16 hours); The next day, inoculate 150mL of LB liquid medium containing Kana (final concentration 200 μg/mL) at a ratio of 1:100, 37'C, Shake the bacteria to 0D _{M at} 220 rpm. =0. 6-1. 0; Then add the inducer IPTG (final concentration ImM) and induce expression at 30' (:, 220 rpm for 6 hours, sample at 0 hours, 2 hours, 4 hours and 6 hours, respectively, to prepare protein Samples were analyzed for expression of foreign protein (TAT-EGFP) by 15% SDS-PAGE and immunoblotting.

The results of the 2 hours and 6 hours test are shown in Fig. 4A and Fig. 4B. It can be seen that the expression of the TAT-EGFP protein (shown by the arrow in the figure) encoded by the foreign plasmid indicates that "competent Escherichia coli BL21 (DE3) "The TAT-EGFP protein encoded by the exogenous plasmid pET28b-Tat-EGFP containing the TAT-EGFP gene can be expressed normally. It can be seen that not only the exogenous substance (the gene encoding the TAT-EGFP protein in this assay) is transferred to the E. coli competent host cell prepared by vacuum freezing, but also the ideal expression is obtained after the exogenous substance is transferred. The TAT-EGFP protein was taken as an example in the assay, indicating that the competent host cell prepared by the present invention can be used as a host cell for gene expression.

Example 2: Preparation of DH5 α competent cells by vacuum freeze-drying technique and rapid transformation of pUC19 As shown in Fig. 5, the exogenous substance was taken as plasmid PUC19 (purchased from THERMO FISHER, 0.5 g/1), and the host cells were Escherichia coli DH5 a is taken as an example. The following steps are the same as in Embodiment 1, and substantially the same expressions may be referred to each other unless otherwise specified.

1) Purification, resuscitation and pretreatment of E. coli host cells (1) Purification of host cells: Escherichia coli host cells were cultured under LS solid medium (formulation: tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar powder 15 g/L) at 37 ° C underlined ( DH5 a ) , obtaining a monoclonal antibody of E. coli host cell DH5 α;

(2) Resuscitation host cells: Pick DH5 _a monoclonal inoculate into 5 mL LB liquid medium (formulation: tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L), 37. C, 220 rpm resuscitation overnight (12-16 hours);

(3) Pre-treatment of host cells: The reconstituted DH5 a bacterial solution was inoculated into 100 mL of fresh LB liquid medium at a ratio of 1:100, 37. (:, rpm at 220 rpm for 3 to 4 hours to ODeo. = 0.6 to 1. 0, divided into 50 ml centrifuge tubes, placed on ice for 10 min cooling, centrifuged at 4000 rpm for 4 min at 10 ° C. Collected bacteria per 50 ml of bacterial solution The weight of the lyophilized protective agent is as follows: The formulation of the lyophilized protective agent is as follows: 1% by weight of gelatin, 7% of mannitol, and the rest is water. According to 200 μl, each tube is dispensed into 1. 5 ml EP tube, -80 °C refrigerator pre-freezing for 12~24 hours or more.

2) Vacuum freeze-drying E. coli host cells

The vacuum freeze-drying instrument (purchased from Beijing Songyuan Huaxing Technology Development Co., Ltd.) was pre-cooled to a temperature of -54 °C and stabilized. The E. coli host cells were quickly removed from the -80'C low temperature refrigerator and placed in a vacuum freeze dryer. In the middle, after installing the cover, close the intake valve and open the vacuum pump, set the -54'C low temperature condition, vacuum freeze the host cells for 12 to 24 hours or more, turn off the vacuum pump after drying, and take out the E. coli treated by the freeze-drying technique. The Escherichia coli prepared by this method is "competent Escherichia coli DH5a", which has the ability to accept the conversion of foreign substances, can be used immediately or stored at -20 °C, or can be stored at 0-4 °C. And transportation.

3) Preparation of an aqueous solution for transforming foreign substances

The pre-transformed plasmid PUC19 (purchased from THERMO FISHER, 0, 5 μg/μ 1 ) was added to 500 1 LB liquid medium (formulation: tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L). The solution was titrated to a concentration of 2 pg/w 1 and kept in a constant temperature water bath at 37 ° C for 30 to 60 minutes.

4) Rapid rehydration of competent E. coli DH5 a, and screening for E. coli host cells transformed with exogenous substances using conditioned medium:

(1) Rapid rehydration of vacuum freeze-dried E. coli host cells: The competent Escherichia coli DH5 a was rapidly added to the LB liquid medium containing plasmid pUC19 which was incubated at 37 ° C, gently mixed and placed. Incubate at 37 rpm in a 37 ° incubator for 45 to 60 minutes;

(2) Screening for E. coli host cells transformed with exogenous substances by conditioned medium pressure: Take 100 μl of competent cells of DH5 a treated with rehydration after freezing and drying, and apply to conditioned medium. LB solid medium containing ampicillin (formulation: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, Agar powder 15g / L, the final concentration of 100μ g / mL of the antibiotic ampicillin), at 37 ° C constant temperature culture for 12 to 16 hours after growing a monoclonal, to obtain a host cell transformed with foreign substances, that is, " pUC19 The transformant ", shown in Figure 5, shows that plasmid pUC19 was successfully transferred into "competent E. coli DH5 alpha".

Example 3. Preparation of Pichia pastoris GS115 competent cells by vacuum freeze-drying technique and rapid transformation of PUC19

The exogenous substance is exemplified by plasmid PUC19 (purchased from THERMO FISHER, 0.5μ _ε /μ 1 ), and the host cell is exemplified by Pichia pastoris GS115. The following steps are the same as those of Embodiment 1 and Embodiment 2, and substantially the same expressions may be referred to each other unless otherwise specified.

1) Purification, resuscitation and pretreatment of E. coli host cells

(1) Purification of host cells: Purification of Pichia pastoris GS115 under 30 Ό with YPD solid medium (formulation: yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L, agar powder 20 g/L) for two days. Yeast cell monoclonal;

(2) Resuscitation host cells: Pick GS115 monoclonal inoculate into 5 mL YPD liquid medium (formulation: yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L), 30. C, 220 rpm resuscitation overnight (12-16 hours);

(3) Pre-treatment of host cells: The reconstituted GS115 bacterial solution was inoculated into 100 mL of fresh YPD liquid medium at a ratio of 1:100 by volume, and shaken at 30 ° C, 220 rpm for 24 to 28 hours to ODe ^ O. 8~1.0, Pipette 50 (^L of bacteria liquid into 1.5mL EP tube, - Pre-freeze in 80 °C refrigerator for 12~24 hours or more.

2) Vacuum freeze-drying Pichia pastoris GS115 host cells

The vacuum freeze-drying instrument (purchased from Beijing Songyuan Huaxing Technology Development Co., Ltd.) was pre-cooled to a temperature of -54 °C and stabilized. The E. coli host cells were quickly removed from the -80 Ό low temperature refrigerator and placed in a vacuum freeze dryer. After installing the lid, close the inlet valve and open the vacuum pump. Set the temperature at -54 °C, freeze and dry the host cells for 12 to 24 hours or more. After drying, turn off the vacuum pump and take out the freeze-dried P. pastoris GS115. The Pichia pastoris GS115 prepared by this method is a "competent yeast" and has the ability to accept the conversion of foreign substances. It can be used immediately or stored at -20'C, or it can be stored at 0-4 °C. transport.

3) Preparation of an aqueous solution for the conversion of exogenous substances

The pre-transformed plasmid UC19 (purchased from THERMO FISHER, .5n g/n 1) was added to 500 μl dd3⁄40 and mixed into an aqueous solution having a concentration of lng/μ1, and kept in a 37'C constant temperature water bath for 30~60. minute.

4) Rapid rehydration of Pichia pastoris GS115 competent cells ("competent yeast") subjected to vacuum freeze-drying, and screening for yeast host cells transformed with exogenous substances by conditioned medium:

(1) Rapid rehydration of vacuum freeze-dried yeast host cells: rapid yeast host cells Add 37°C incubation-containing aqueous solution containing plasmid pUC19 (equal volume to host cells before freeze-drying), mix gently, incubate at 37 ° C for 30 minutes, and then incubate in a 30 ° C incubator at 220 rpm. 1 to 4 hours;

(2) screening the yeast host cells transformed with the foreign substance by conditioned medium: 100 μl of the yeast host cell solution after lyophilization and rehydration treatment is applied to the conditioned medium containing ampicillin. YPD solid medium (formulation: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agar powder 20g / L, final concentration 100 μg / mL antibiotic ampicillin), constant temperature culture at 30 2 2 After 3 days, a monoclonal antibody is grown to obtain a host cell into which a foreign substance has been transformed, that is, " _P UC19 yeast transformed bacteria". Referring to Fig. 6, it was shown that plasmid pUC19 was successfully transferred into "competent yeast", and yeast competent cells were prepared and the transfer of exogenous substance PUC19 was achieved.

Industrial applicability

The invention utilizes the vacuum freeze-drying technology to prepare the competent host cells, and can rapidly and efficiently transfer foreign substances such as nucleic acids, proteins, amino acids and sugars into the competent host cells, and the method has the advantages of simple operation, small damage, low cost and the like. advantage. Through the implementation of the present invention, not only a batch of competent host cells can be obtained simply and quickly, but also can be used for subsequent biological function research, and can also provide an important technical means for transmembrane transport of foreign substances, and the invention has industrial application. prospect.

Claims

Rights request

1. A method for preparing competent host cells, including the following steps:

1) Purification, recovery and pretreatment of host cells;

2) Use vacuum freeze-drying technology to treat host cells to obtain competent host cells.

2. The method according to claim 1, characterized in that: the host cell is any prokaryotic or eukaryotic host cell, including bacterial cells, fungal cells, plant cells and animal cells, such as E. coli and yeast.

3. The method according to claim 1 or 2, characterized in that: Step 1) The purified host cells refer to streaking and culturing the original host cells on the surface of a solid medium to obtain a single clone; reviving the host cells refers to purifying the host cells. The host cells are re-cultured for a certain period of time to re-enter the growth state from the resting state; pretreatment. I cells refer to the expanded culture of the recovered host cells to the corresponding concentration.

4. The method according to claim 3, characterized in that: Step 1) The process of purifying, resuscitating and pretreating host cells is as follows:

(1) Purify host cells: Use an inoculating loop to take the original host cell partitions and inoculate them into a solid medium suitable for cultivating host cells, and culture them under appropriate conditions to obtain a single clone of the host cell: LB for E. coli Solid culture medium, its formula is tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar powder 15g/L _; YPD solid culture medium for yeast, its formula is yeast extract 10g/L, peptone 20g /L, glucose 20g/L, agar powder 20g/L; suitable culture conditions for E. coli are 37°C, 12~16h; suitable culture conditions for yeast are 30°C, 48h _;

(2) Resuscitation of host cells: Pick a single clone of host cells and inoculate it into 5 mL of growth medium suitable for host cells, and recover at a suitable temperature (E. coli 37'C _; yeast culture 30'C) 220r _P m overnight (12 ~16 hours); LB medium suitable for E. coli, its formula is tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L; YPD medium suitable for yeast, its formula is 10g yeast extract /L, peptone 20g/L, glucose 20g/L;

(3) Pretreatment of host cells: Inoculate the revived host cells into 100 mL of the fresh growth medium described in step (2) at a volume ratio of 1:100, and culture at an appropriate temperature (E. coli 37Ό; yeast culture 30 °C) Shake at 220rpm to

6~1.0; Pipette 500μ1 bacterial liquid into 1.5mL EP tube, and pre-freeze in 80°C refrigerator for more than 12~24 hours.

5. The method according to any one of claims 1 to 4, characterized in that: the vacuum freezing and drying technology treatment in step 2) is to pre-cool the host cells treated in step 1) with a vacuum freeze dryer for 30~ Wait for 120 minutes until the temperature reaches -54. C and stabilize at this temperature, then vacuum freeze-dry the host cells for 12 to 24 hours or more.

6. A competent host cell prepared by the method of any one of claims 1 to 5, and the competent host cell still possesses biological activity at a temperature of 0-4°C.

7. A method for transferring exogenous substances into host cells, using the competent host cells prepared by the method described in any one of claims 1 to 5, further comprising the following steps:

3) Prepare an aqueous solution of the exogenous substance to be transformed;

4) Use the aqueous solution to quickly rehydrate the competent host cells obtained in step 2) to obtain a host cell bacterial solution that has been transformed with exogenous substances;

In this way, foreign substances are transferred into the host cells.

8. The method according to claim 7, characterized in that the exogenous substances used include nucleic acids (deoxyribonucleic acid (DNA), ribonucleic acid (RNA)), proteins, oligopeptides, amino acids and other biologically active substances.

9. The method according to claim 7 or 8, characterized in that, the exogenous substance aqueous solution in step 3) uses ddH ₂ 0, and the prepared exogenous substance aqueous solution is kept in a 37°C water bath for 30~ 60 minutes.

10. The method according to claim 7 or 8 or 9, characterized in that the operation of rapid rehydration in step 4) is: rapidly adding the competent host cells in step 2) to the aqueous solution of the exogenous substance prepared in step 3), Mix gently and then place in an incubator at a suitable temperature (30'C for E. coli yeast culture) with shaking at 220 rpm for 45 to 60 minutes; the amount of aqueous solution of exogenous substances should be the same volume as the host cells before freeze-drying.

11. A method for generating exogenous substance-transformed bacteria, characterized in that the host cells transformed with exogenous substances obtained by the method of any one of claims 7 to 10 are screened using conditioned medium, and the obtained single clone is Bacteria transformed by foreign substances.

12. The method according to claim 11, characterized in that the conditioned medium suitable for Escherichia coli is LB solid medium, and the formula is: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar Powder 15g/L, final concentration 100-200 g/mL of antibiotics suitable for exogenous substances; suitable for yeast is YPD solid medium, the formula is: yeast extract 10g/L, peptone 20g/L, glucose 20g/L, Agar powder 20g/L, final concentration 100-200 μg/mL suitable for antibiotics of exogenous substances.

13. The method according to claim 11 or 12, characterized in that the screening process is: inoculating or coating the host cell bacterial liquid transformed with the exogenous substance into the conditioned medium, at a suitable temperature (E. coli 37 ; Yeast culture at 30°C) After culturing at a constant temperature for 12 to 16 hours, a single colony grows, and the exogenous substance transformed bacteria are obtained.

14. The exogenous substance transformed bacteria prepared by the method of any one of claims 11 to 13.