WO2019140926A1

WO2019140926A1 - Functional bio-nano-magnetic bead fluorescence coding method and flow application thereof

Info

Publication number: WO2019140926A1
Application number: PCT/CN2018/104214
Authority: WO
Inventors: 张金菊; 王红光
Original assignee: 北京国科融智生物技术有限公司
Priority date: 2018-01-22
Filing date: 2018-09-05
Publication date: 2019-07-25
Also published as: CN108387729A; CN108387729B

Abstract

Disclosed is a functional bio-nano-magnetic bead fluorescence coding method and a flow application thereof. The membrane protein of the magnetic bead allows simultaneous fusion expression of a fluorescent protein and a functional protein, can uniformly express and exhibit the fluorescent protein on the surface, and can be directly used as a fluorescent labeled bio-nano-magnetic bead. The surface of the bio-nano-magnetic bead can also simultaneously express and exhibit protein molecules having other bioactivity, such as recombinant proteins, to meet the need for flow detection.

Description

Functional biological nano magnetic bead fluorescence coding method and its flow application

Technical field

The invention belongs to the field of biological nano magnetic beads application and medical inspection technology, and particularly relates to a functional biological nano magnetic bead fluorescence coding method and a flow application thereof.

Background technique

Bio-nanomagnetic beads are magnetic nanoparticles produced by magnetotactic bacteria, also known as bacterial magnetic particles. The inner core is Fe ₃ O ₄ crystal, which is coated with a layer of phospholipid biofilm and has a particle size of 30-120 nm. The bio-nanomagnetic beads produced by the same kind of magnetotactic bacteria have the same particle size and crystal crystal form, uniform magnetic properties, natural biofilm coating, and good water-soluble and colloidal properties. In addition, bacterial magnetic particles are a source of biological preparation and therefore have good biocompatibility. The surface of the bio-nanomagnetic bead has a large number of amino groups, which can be linked to different functional macromolecules, such as antibodies, by chemical modification and bifunctional coupling agents, thereby having different special functions. The most unique feature of bacterial magnetic particles is that they can express specific protein and polypeptide molecules on the surface membrane by genetic engineering methods, and become functional biological nano-magnetic beads with special biological activity.

Fluorescence coding technology is a new nanotechnology that has emerged since 2000. By combining nanometer quantum dots with different numbers and different fluorescence characteristics, fluorescent microspheres are prepared. The microspheres have unique fluorescence coding features and can be The identification and differentiation of optical analysis equipment has a strong application prospect in flow cytometry. In theory, only 5-6 fluorescent colors and quantum dots of 5-6 fluorescence intensities are required, and more than 30,000 different combinations of fluorescently encoded microspheres can be combined. In terms of application, the basic design of fluorescent coded microspheres is to connect different biomolecules on the surface of the microspheres, and design different fluorescent coded signals inside the microspheres to finally achieve simultaneous quantitative detection of more than 30,000 target substances in the liquid phase. analysis. Although the currently applied fluorescent coded microspheres can only detect tens to hundreds of targets at the same time, it is still far from the theoretical value, but the efficiency of liquid phase biomolecule detection has been greatly improved.

Fluorescent protein is a fluorescent protein substance isolated from organisms. It was the first green fluorescent protein found in jellyfish in 1962. It has been widely used in fluorescent science in the field of life sciences. The development of technology has also indirectly promoted the development of fluorescent nanomaterials. The 2008 Nobel Prize in Chemistry was awarded to three scientists who have made outstanding contributions to the discovery and research of fluorescent proteins. Through the use of a series of theories and techniques such as bioinformatics, gene mutation, and DNA-shuffling engineering, the members of the fluorescent protein family have expanded, and many new fluorescent protein mutants have appeared, which have different spectral characteristics. Representative fluorescent proteins are available in five colors: green, cyan, blue, yellow, and red. These different fluorescent protein mutants further extend the range of applications for fluorescent labeling.

technical problem

In recent years, the assembly of nanomaterials and fluorescent materials has emerged to form new functional nanomaterials, which have great potential in the field of biomedicine. The fluorescent substances here are mostly inorganic fluorescent materials or organic small molecule fluorescent dyes. Most of the methods are also chemical coupling methods. The invention mainly provides a functional biological nano magnetic bead from the viewpoint of a biosynthesis method and a fluorescent protein, wherein a fluorescent protein is displayed on the surface, and the production of the fluorescently encoded magnetic nanoparticle is realized by selecting different fluorescent protein combinations.

Technical solution

In order to solve the above technical problems, the present invention provides a functional biological nano magnetic bead fluorescence encoding method and its streaming application.

The specific technical solutions of the present invention are as follows:

In one aspect, the invention provides a functional bio-nanomagnetic bead, wherein the membrane protein of the functional bio-nanomagnetic bead is simultaneously fused to express a fluorescent protein and a functional protein.

The functional bio-nanomagnetic beads provided by the invention can be directly used as fluorescent labeled bio-nano magnetic beads, and the functional proteins expressed by the flow cytometer can be detected.

Further, the fluorescent protein is any one of EGFP, EBFP, ECFP, EYFP, ERFP, mOrange, mCherry, mStrawberry, mRaspberry, mPlum, and mKate, and the EGFP gene sequence is as shown in SEQ. The EBFP gene sequence is shown in SEQ. ID. No. 2, the ECFP gene sequence is shown in SEQ. ID. No. 3, and the EYFP gene sequence is shown in SEQ. ID. No. The ERFP gene sequence is shown in SEQ. ID. No. 5, the mOrange gene sequence is shown in SEQ. ID. No. 6, and the mCherry gene sequence is shown in SEQ. ID. No. 7, the mStrawberry The gene sequence is shown in SEQ. ID. No. 8, the mRaspberry gene sequence is shown in SEQ. ID. No. 9, and the mPlum gene sequence is shown in SEQ. ID. No. 10, the mKate gene sequence. As shown in SEQ.ID.No.11.

The above fluorescent proteins are all improved enhanced fluorescent proteins, and the sensitivity of flow detection is higher and the accuracy is better.

Further, the functional protein is recombinant protein G, recombinant protein A or recombinant streptavidin protein SA, and the gene sequence of the recombinant protein G is shown in SEQ. ID. No. 12, and the recombinant protein A is The gene sequence is shown in SEQ. ID. No. 13, and the gene sequence of the recombinant streptavidin protein A is shown in SEQ.

Another aspect of the present invention provides a method of fluorescently encoding the above-described functional biological nanomagnetic beads, comprising the steps of:

S1: Using a magnetotactic bacteria MSR-I to construct a deletion mutant strain of a bacterial magnetic particle membrane protein gene, as a primary recombinant strain, the bacterial magnetic particle membrane protein gene deleted is any two of MamC, MamD and MamF Gene

S2: using any of the deleted bacterial magnetic particle membrane protein gene fusion functional proteins, and introducing into the primary recombinant strain to construct a secondary recombinant strain;

S3: constructing a gene fusion expression vector by using the bacterial magnetic particle membrane protein gene fusion fluorescent protein gene deleted in any one of steps different from step S2; introducing the gene fusion expression vector into the secondary recombinant strain, and Screening to obtain a three-stage recombinant strain having fluorescent coding properties;

S4: The third-stage recombinant strain is cultured to obtain a functional biological nano-magnetic bead capable of simultaneously expressing a fluorescent protein and a functional protein.

The bio-nano magnetic beads obtained by the method can uniformly express the fluorescent protein on the surface and can be directly used as the fluorescent labeled bio-nano magnetic beads; meanwhile, the surface of the bio-nano magnetic beads can simultaneously express and display other biological activities such as recombinant protein. Protein peptides to meet the needs of flow detection.

Further, the step S1 includes the following steps:

S1.1: amplifying a 500 bp homologous DNA fragment flanking the two membrane protein genes, respectively, and constructing two microcarriers by molecular cloning;

S1.2: transferring the two microcarriers into the MSR-I wild type strain simultaneously by electroporation;

S1.3: Screening and identification of the electrotransformed strain to obtain a recombinant strain lacking two kinds, which is a primary recombinant strain.

In the present invention, the use of phage AAV-del microcarriers is mentioned. The difference between the conventional plasmids is that the plasmids can exist independently of the chromosomes, can replicate themselves, can exist in the cells for a long time, and are not easily lost; the microcarriers cannot be in the chromosomes. Self-replication alone, only integrated into the chromosome can exist for a long time and is easy to lose. Therefore, it is used for gene knockout, the genetic background is relatively clean, and there is not much interference and pollution of foreign genes.

Further, the step S2 includes the following steps:

S2.1: fusing the gene sequence of the functional protein with a deleted sequence of the bacterial magnetic particle membrane protein gene to obtain a new fusion gene fragment;

S2.2: cloning the fusion gene fragment into the expression vector pBRC1 to obtain a functional protein expression plasmid;

S2.3: transferring the functional protein expression plasmid into the primary recombinant strain by means of triple parental ligation or electroporation, and obtaining a recombinant strain expressing the functional protein after verification, that is, a secondary recombinant strain.

Further, the method of step S3 is as follows:

S3.1: performing PCR amplification on the fluorescent protein gene sequence;

S3.2: Double digestion with the amplification product and the expression vector pBRC2 by EcoRI/SmaI;

S3.3: the double-digested amplification product is fused with the bacterial magnetic particle membrane protein gene different from the deletion of step S2, and ligated to the expression vector pBRC2 which is also double-digested. Fluorescent protein expression plasmid;

S3.4: 11 fluorescent protein fusion expression plasmids were separately transformed into the secondary recombinant strain by electroporation, and the tertiary recombinant strain capable of expressing fluorescent protein was obtained through screening and verification;

S3.5: The different fluorescent proteins expressed by the tertiary recombinant strain were analyzed and identified by flow cytometry. The emission peaks of different fluorescent proteins were as follows: EGFP: 503-506 nm; EBFP: 446-450 nm; EYFP: 523 ~526nm; ECFP: 475~477nm; ERFP: 607~609nm; mOrange: 557~561nm; mCherry: 615nm; mStrawberry: 574~577nm; mRaspberry: 621~625nm; mPlum: 646~649nm; mKate: 633~636nm.

Further, the step S4 includes the following steps:

S4.1: Firstly, using 200~500mL medium for pre-culture, the culture condition is oxygen content 5~10%, N2 content 90~95%, culture time 16 hours, culture temperature 37 °C;

S4.2: transferring the pre-cultured strain to the fermenter for deep culture, the culture condition is 5% oxygen content, hydrogen content 1%, N2 content 94%, culture time 3-4 days, culture temperature 37 ° C;

S4.3: treating the deep culture by homogenizing equipment, pulverizing the bacteria, adsorbing the magnetic particles of the bacteria through a magnetic device, and washing with the phosphate buffer for 2 to 3 times;

S4.4: Gradient treatment with ultrasonic and protease buffer to finally obtain purified functionalized magnetic particles of the bacteria.

The MSR–I strain is a microaerobic bacterium, which is easy to absorb external iron ions to synthesize nano-magnetic beads in a micro-aerobic environment, and the synthesis of nano-magnetic beads is greatly reduced under conditions of high oxygen or oxygen, so fermentation culture The oxygen content should be precisely controlled during the process to increase the yield of the nanomagnetic beads. At the same time, it was found in this study that increasing the hydrogen content is also beneficial to increase the yield of nanomagnetic beads.

Further, the specific method of the electrical conversion is as follows:

Using square wave electric pulse, the electric pulse is 3.1~3.3ms in 1~2 times under the condition of 3100~3200V.

Gene transfer is usually carried out by means of conjugation between bacteria, which easily causes loss of the transferred gene. The way of electroporation is to form a hole in the cell by instantaneous current, so that the DNA gene fragment in the solution can enter the cell through the hole, thereby completing the transformation. When the current of electroconversion is too small, pores can be formed or pores are formed for a short period of time, which is not conducive to gene transfer; if the current is too large and the time is too long, it is easy to cause irreversible damage to the cells and even cause cell death. The above electrotransformation conditions can effectively transfer the microcarrier or the expression plasmid into the recipient cell without causing loss of the gene fragment or causing irreversible damage to the cell activity.

Further, the medium used in the pre-culture includes the following components:

10~12 parts beef extract, 1~2 parts chitosan, 1~2 parts galactose, 0.5~1 part sodium dihydrogen phosphate, 0.2~0.5 parts phosphatidylserine, 0.05~0.1 parts sodium citrate and 0.5~ 1 part potassium alginate;

The medium used in the deep culture includes the following components:

6~8 parts of Ligustrum lucidum polysaccharide, 8~12 parts of peptone, 1~2 parts of snail polysaccharide, 1~2 parts of lecithin, 0.1~0.2 parts of quercetin, 0.5~1 part of sodium potassium tartrate and 0.05~0.1 parts of lemon Sodium.

Pre-culture of the third-stage recombinant strain using the above medium can effectively increase the activity of the bacteria, thereby improving the proliferation ability and the viability of the subsequent culture; and using the above medium to deeply culture the pre-cultured tertiary recombinant strain, It can effectively improve the resistance of bacteria and the carrying capacity of fluorescent protein fusion expression plasmids, promote the proliferation of bacteria and the simultaneous expression of fluorescent proteins and recombinant proteins, thus facilitating subsequent flow analysis.

Beneficial effect

The beneficial effects of the present invention are as follows: The present invention provides a functional biological nano-magnetic bead fluorescence encoding method and a flow application thereof, and the biological nano magnetic beads obtained by the method can uniformly express the fluorescent protein on the surface, and can be directly used as Fluorescently labeled bio-nanomagnetic beads are used; at the same time, the surface of the bio-nanomagnetic beads can simultaneously express other biologically active protein molecules such as recombinant proteins to meet the needs of flow detection. Using the medium provided by the method to deeply culture the tertiary recombinant strain can effectively improve the activity and stress resistance of the bacteria, and enhance the carrying capacity of the bacterial fluorescent protein fusion expression plasmid, promote bacterial proliferation, and fluorescent protein and recombination. Simultaneous expression of the protein ensures the quality and yield of functional bio-nanobead synthesis, facilitating subsequent fluorescence-encoded flow analysis applications.

DRAWINGS

1 is a flow chart of a blank control in Experimental Example 1;

2 is a flow chart of a negative control in Experimental Example 1;

3 is a flow chart of Experimental Group 1 in Experimental Example 1;

4 is a flow chart of Experimental Group 2 in Experimental Example 1;

5 is a flow chart of Experimental Group 3 in Experimental Example 1;

6 is a flow chart of Experimental Group 4 in Experimental Example 1;

Fig. 7 is a graph showing the fluorescence intensity decay curves of the biomagnetic nanobeads of each group in Experimental Example 7.

Embodiments of the invention

Example 1

A functional biological nanomagnetic bead expressing EGFP fluorescent protein, which expresses recombinant protein G on MamF membrane protein, and simultaneously expresses fluorescent protein EGFP on MamC membrane protein.

Example 2

A functional biological nanomagnetic bead expressing EBFP fluorescent protein, which expresses recombinant protein A on MamF membrane protein, and simultaneously expresses fluorescent protein EBFP on MamC membrane protein.

Example 3

A functional bio-nanomagnetic bead expressing an ECFP fluorescent protein, which expresses a recombinant chain affinity protein SA on a MamF membrane protein, and simultaneously expresses a fluorescent protein ECFP on a MamC membrane protein.

Example 4

A functional bio-nanomagnetic bead expressing EYFP fluorescent protein, which expresses recombinant protein G on MamF membrane protein, and simultaneously expresses fluorescent protein EYFP on MamC membrane protein.

Example 5

A functional biological nanobead fluorescent coding method for expressing ERFP fluorescent protein, comprising the following steps:

S1: using the magnetotactic bacteria MSR-I to construct a deletion mutant strain of the bacterial magnetic particle membrane protein genes MamC and MamF as a primary recombinant strain;

S2: using the MamF gene to fuse the recombinant protein G, and introducing into the primary recombinant strain to construct a secondary recombinant strain;

S3: using the MamC gene to fuse the ERFP fluorescent protein gene, constructing an expression vector, introducing the expression vector into the secondary recombinant strain, and screening to obtain a tertiary recombinant strain having fluorescent coding characteristics;

S4: The tertiary recombinant strain is cultured to obtain functional biological nanomagnetic beads capable of simultaneously expressing the fluorescent protein ERFP and the recombinant protein G.

Example 6

A functional biological nanobead fluorescent coding method for expressing mOrange fluorescent protein, comprising the following steps:

S1.1: Amplification of a 500 bp homologous DNA fragment flanking the MamC gene and the MamF gene, and constructing two microcarriers AAV-del-MamC and AAV-del-MamF by molecular cloning;

S1.2: AAV-del-MamC and AAV-del-MamF were mixed at a molar ratio of 2:1 to a final concentration of 2 mg/mL, and simultaneously transferred into the MSR-I wild-type strain by electroporation;

S1.3: The electrotransformed strain is screened for double mutant strains by sucrose and antibiotic gradient concentration pressure. The method of electrotransformation is to use square wave electric pulse, and the time is 3~3 times and the length is 3.1~3.3ms under the condition of 3100~3200V. Electric pulse; after identification of the strain, the recombinant strain with MamC and MamF double deletion is obtained, which is the primary recombinant strain MSRI-dCF;

S2: using MamF gene fusion recombinant protein A, and introducing into a primary recombinant strain to construct a secondary recombinant strain;

S3: using the MamC gene to fuse the mOrange fluorescent protein gene, constructing an expression vector, introducing the expression vector into the secondary recombinant strain, and screening to obtain a tertiary recombinant strain having fluorescent coding characteristics;

S4: The tertiary recombinant strain is cultured to obtain a functional biological nanomagnetic bead capable of simultaneously expressing the fluorescent protein mOrange and the recombinant protein A.

Example 7

A functional biological nanobead fluorescent coding method for expressing mStrawberry fluorescent protein, comprising the following steps:

S2: using the MamF gene to fuse the recombinant affinity protein SA, and introducing into the primary recombinant strain to construct a secondary recombinant strain;

S2.1: The gene sequence of the recombinant strand affinity protein SA is fused with the MamF gene sequence to obtain a new fusion gene fragment pMamF-SA;

S2.2: The new fusion gene fragment was cloned into the expression vector pBRC1 to obtain the expression plasmid pBRC1-pMamF-SA;

S2.3: Transfer the expression plasmid into the primary recombinant strain by electroporation. The method of electrotransformation is to use a square wave electric pulse to perform 1~2 times of 3.1~3.3ms electric pulse at 3100~3200V. After verification, a secondary recombinant strain MSRI-dCF/SA expressing recombinant strand affinity protein SA was obtained;

S3: using the MamC gene fusion mStrawberry fluorescent protein gene, constructing an expression vector, introducing the expression vector into the secondary recombinant strain, and screening to obtain a tertiary recombinant strain having fluorescent coding characteristics;

S4: The tertiary recombinant strain was cultured to obtain a functional bio-nanomagnetic bead capable of simultaneously expressing the fluorescent protein mStrawberry and the recombinant streptavidin SA.

Example 8

A functional biological nanobead fluorescent coding method for expressing mCherry fluorescent protein, comprising the following steps:

S3: using the MamC gene to fuse the mCherry fluorescent protein gene to construct an expression vector;

S3.1: PCR amplification of the mCherry gene;

S3.3: the double-digested amplification product is fused with the MamC gene, and ligated to the expression vector pBRC2 which has also been double-digested to obtain a fluorescent protein expression plasmid pBRC-mCherry of the fusion MamC;

S3.4: The fluorescent protein fusion expression plasmid pBRC-mCherry is transformed into the secondary recombinant strain by electroporation, and the electrotransformation method is performed by using a square wave electric pulse at 1100 to 3200 V for 1 to 2 times. The duration is 3.1~3.3ms electric pulse; the third-stage recombinant strain capable of expressing mCherry is obtained through screening and verification;

S3.5: analysis and identification of mCherry expressed by the tertiary recombinant strain by flow cytometry at a wavelength of 523-526 nm;

S4: The tertiary recombinant strain is cultured to obtain a functional biological nanomagnetic bead capable of simultaneously expressing the fluorescent protein mCherry and the recombinant protein G.

Example 9

A functional biological nanobead fluorescent coding method for expressing mRaspberry fluorescent protein, comprising the following steps:

S3: using the MamC gene fusion mRaspberry fluorescent protein gene to construct an expression vector; introducing the expression vector into the secondary recombinant strain, and screening to obtain a tertiary recombinant strain having fluorescent coding characteristics;

S4: cultivating the third-stage recombinant strain to obtain a functional biological nano-magnetic bead capable of simultaneously expressing the fluorescent protein mRaspberry and the recombinant protein A;

S4.1: Firstly, using 200~500mL medium for pre-culture, the culture condition is micro-aerobic (O ₂ content 5~10%, N ₂ content 90~95%), culture time 16 hours, culture temperature 37 ° C;

S4.2: Transfer the pre-cultured strain to a fermenter for deep culture, and the culture condition is micro-aerobic plus hydrogen (O ₂ content 5%, H ₂ content 1%, N ₂ content 94%), culture time 3~4 days, culture temperature 37 °C;

Example 10

A method for fluorescently encoding a functional biological nanomagnetic bead expressing mPlum fluorescent protein is the same as the step of Example 9, wherein the medium pre-cultured in step S4 comprises the following components:

12 parts of beef extract, 1 part of chitosan, 2 parts of galactose, 0.5 part of sodium dihydrogen phosphate, 0.5 part of phosphatidylserine, 0.1 part of sodium citrate and 0.5 part of potassium alginate;

The medium for deep culture includes the following ingredients:

8 parts of Ligustrum lucidum polysaccharide, 8 parts of peptone, 1 part of snail polysaccharide, 2 parts of lecithin, 0.2 part of quercetin, 0.5 part of sodium potassium tartrate and 0.1 part of sodium citrate.

Comparative Example 1

A method for fluorescently encoding a functional biological nanomagnetic bead expressing mOrange fluorescent protein is the same as the procedure of Example 6, wherein the bacterial magnetic particle membrane protein genes deleted by the primary recombinant strain are MamC and MamD.

Comparative Example 2

A method for fluorescently encoding a functional biological nanomagnetic bead expressing mOrange fluorescent protein is the same as the procedure of Example 6, wherein the bacterial magnetic particle membrane protein genes deleted by the primary recombinant strain are MamD and MamF.

Comparative Example 3

A method for fluorescently encoding a functional biological nanomagnetic bead expressing mOrange fluorescent protein is the same as the procedure of Example 6, wherein the bacterial magnetic particle membrane protein gene deleted by the primary recombinant strain is MamA and MamC.

Comparative Example 4

A functional biological nanomagnetic bead fluorescent coding method for expressing mOrange fluorescent protein is the same as the procedure of Example 6, wherein the molar ratio of the two microcarriers AAV-del-MamC and AAV-del-MamF is 1:1.

Comparative Example 5

A method for fluorescently encoding a functional biological nano-magnetic bead expressing mcherry fluorescent protein is the same as the procedure of Example 8, wherein the method of electrotransformation is performed by using a square wave electric pulse at a temperature of 2700 to 2800 V for 1 to 2 times and a duration of 3.1. ~3.3ms electrical pulse.

Comparative Example 6

A functional biological nano-magnetic bead fluorescence coding method for expressing mcherry fluorescent protein is the same as the method of the eighth embodiment, wherein the electrotransformation method is performed by using a square wave electric pulse, and the 1-2 time period is 2.8 at 3100~3200V. ~3.0ms electric pulse.

Comparative Example 7

A functional biological nano-magnetic bead fluorescence coding method for expressing mcherry fluorescent protein is the same as the method of the eighth embodiment, wherein the electrotransformation method is performed by using a square wave electric pulse, and the time is 3 to 3 times at a temperature of 3300 to 3400 V. ~3.3ms electrical pulse.

Comparative Example 8

A functional biological nano-magnetic bead fluorescence coding method for expressing mcherry fluorescent protein is the same as the procedure of the eighth embodiment, wherein the electrotransformation method is performed by using a square wave electric pulse, and the time is 3.4 times at 3100~3200V for 1-2 times. ~3.6ms electrical pulse.

Comparative Example 9

A functional biological nanobead fluorescent coding method for expressing mRasperry fluorescent protein is the same as the procedure of Example 9, wherein the pre-culture conditions are an O ₂ content of 15% and an N ₂ content of 85%.

Comparative Example 10

A functional biological magnetic bead fluorescence coding method mRasperry fluorescent protein expressed, in Example 9, Step, submerged culture conditions in which the O ₂ content is 5%, N ₂ content of 95%.

Comparative Example 11

A functional biological nanobead fluorescent coding method for expressing mRasperry fluorescent protein is the same as the procedure of Example 9, wherein the conditions of the deep culture are 10% of O ₂ content, 1% of H ₂ content, and 89% of N ₂ content.

Comparative Example 12

A method for fluorescently encoding a functional biological nanomagnetic bead expressing mPlum fluorescent protein is the same as the step of Example 10, wherein the medium for pre-culture and sub-culture in step S4 is LB medium.

Comparative Example 13

A method for fluorescently encoding a functional biological nanomagnetic bead expressing mPlum fluorescent protein, which is the same as the step of Example 10, wherein the medium pre-cultured in step S4 is the medium provided in Example 6, and the medium cultured in deep culture is wort. Medium.

Comparative Example 14

A method for fluorescently encoding a functional biological nanomagnetic bead expressing mPlum fluorescent protein, which is the same as the step of Example 10, wherein the medium pre-cultured in step S4 is LB medium, and the medium cultured in deep culture is cultured in Example 6. base.

Comparative Example 15

A functional biological nanomagnetic bead expressing EGFP fluorescent protein, which expresses the fluorescent protein EGFP on the MamC membrane protein.

Comparative Example 16

A functional bio-nanomagnetic bead expressing EYFP fluorescent protein, which expresses the fluorescent protein EYFP on the MamC membrane protein.

Experimental example 1

Fluorescence-coded biological nano magnetic beads flow analysis experiment

The cultured CHO cells were used as the detection object, and the bio-nanomagnetic beads provided in Examples 1 to 4 were used as the experimental groups 1 to 4, and the recombinant protein G biological nanomagnetic beads without fluorescence coding were used as the negative control to be untreated. CHO cells were used as blank controls.

The above five bio-nanomagnetic beads were respectively combined with an anti-CHO antibody, and incubated at room temperature for 15 min, and the related IgG antibody was bound by the recombinant protein G, and coupled to the bio-nano magnetic beads; a five-flow analysis tube was taken. Add about 10 ⁵ CHO cells to each tube, resuspend them evenly, add 50 μL of five bio-nanomagnetic beads labeled with -CHO antibody, incubate for 15 min at room temperature, mix 2 to 3 times, and wash the cells by magnetic device. The impurities and unbound cells were removed; the above cell samples were tested using a flow cytometer.

The experimental results are shown in Figures 1 to 6. The negative control did not produce fluorescence excitation, which was consistent with the blank control results. However, the spectra of the experimental groups changed, and the peak times were different, indicating that the fluorescent coded biological nano magnetic in 4 The corresponding fluorescence excitation was produced after the beads bound CHO cells. It is thus illustrated that the bio-nanomagnetic beads provided by the present invention can simultaneously express fluorescent proteins and recombinant proteins, thereby facilitating subsequent flow analysis.

Experimental example 2

Membrane protein gene deletion comparison experiment

The methods provided in Examples 5 to 6 were used as the experimental groups 1 to 2, and the methods provided in the comparative examples 1 to 4 were used as the control groups 1 to 4, and the magnetotactic bacteria MSR-I was cultured by the above method to the wild type strain. As a positive control, the magnetic bead yield of each group of bacteria was measured and compared. The experimental results are shown in Table 1.

Table 1 Production of nanomagnetic beads per liter of culture medium in each group of cultures

组别Group	纳米磁珠含量（mg）Nano magnetic beads content (mg)	占比（%）Proportion (%)
阳性对照Positive control	256.2256.2	11
实验组1Experimental group 1	226.7226.7	88.588.5
实验组2Experimental group 2	229.4229.4	89.589.5
对照组1Control group 1	156.3156.3	61.061.0
对照组2Control group 2	147.6147.6	57.657.6
对照组3Control group 3	98.698.6	38.538.5
对照组4Control group 4	175.3175.3	68.468.4

It can be seen from Table 1 that the magnetic bead yield of each group in the experimental group can reach more than 85% of the wild type, and the decrease is not obvious; while the magnetic bead yield of each group in the control group is significantly lower than that of the wild type strain, among which the control group The magnetic beads yield of 3 and 4 is the lowest. It is indicated that MamC+MamF is the optimal choice when constructing a membrane protein double gene-depleted magnetotactic bacteria; and when the same deletion membrane protein is selected, it is used to bind to express fluorescent protein and to bind to express functional protein. When the molar ratio of the microcarriers is 2:1, the effect is better than 1:1.

Experimental example 3

Electroporation conditions comparison experiment

The method provided in Example 8 was used as the experimental group 1, and the methods provided in the comparative examples 5 to 8 were used as the control groups 1 to 4, and the magnetotactic bacteria MSR-I was cultured by the above method, and the number of bacteria in each group was 107. Two 1 μg (about 5 kbp in size) of the transformed DNA was used to measure and compare the survival rate of the electrotransformed bacteria and the success rate of gene expression expression. The experimental results are shown in Table 2.

Table 2 Survival rate and conversion success rate after electrotransformation of each group of bacteria

组别Group	细菌成活率（%）Bacterial survival rate (%)	转化成功率（%）Conversion success rate (%)
实验组1Experimental group 1	72.872.8	38.338.3
对照组1Control group 1	71.471.4	17.217.2
对照组2Control group 2	68.768.7	19.519.5
对照组3Control group 3	51.151.1	22.322.3
对照组4Control group 4	48.648.6	20.920.9

It can be seen from Table 2 that the survival rate and gene conversion success rate of the experimental group were significantly higher than those of the control group, among which the survival rate of the control group 1 and 2 was higher, but the conversion success rate was lower, and the second control group 3, 4 The survival rate and conversion success rate are both low. It indicates that the current is too low or the time is too short, which will reduce the success rate of the conversion. If the current is too large or too long, the bacterial mortality will increase, which will also affect the success rate of the conversion; thus indicating the electrotransformation conditions provided by the present invention. The success rate of conversion can be improved under the premise of ensuring the survival rate of bacteria.

Experimental example 4

Fermentation culture condition comparison experiment

The method provided in Example 9 was used as the experimental group 1, and the methods provided in the comparative examples 9 to 11 were used as the control group 1 to 3. The magnetotactic bacteria MSR-I was cultured by the above method, and the wild type strain was used as a positive control. The magnetic bead yield of each group of bacteria was measured and compared. The experimental results are shown in Table 3.

Table 3 Production of nanomagnetic beads per liter of medium in each group of cultures

组别Group	纳米磁珠含量（mg）Nano magnetic beads content (mg)	对照组减产比例（%）Reduced production ratio of the control group (%)
实验组1Experimental group 1	232.5232.5	—-
对照组1Control group 1	177.2177.2	23.823.8
对照组2Control group 2	192.7192.7	17.117.1
对照组3Control group 3	171.6171.6	26.226.2

As can be seen from Table 3, the magnetic bead yield of the experimental group was significantly higher than that of the control group, and the production of the magnetic beads of the control group 1 and 3 was significantly lower than that of the control group 2. It is indicated that the culture conditions of micro-aerobic + small amount of hydrogen provided by the present invention can significantly increase the magnetic bead yield of the magnetotactic bacteria MSR-I.

Experimental example 5

Medium performance comparison test

Under the same conditions of inoculum, the method provided in Example 10 was used as Experimental Example 1, and the methods provided in Comparative Examples 12 to 14 were used as Comparative Examples 1 to 3 to culture the magnetotactic bacteria MSR-1, respectively. The bacterial viability and quantity were determined and compared. The experimental results are shown in Table 4.

Table 4 Vigor and quantity of bacteria in each group

组别Group	细菌活力Bacterial vigor	细菌数量Number of bacteria
实验组1Experimental group 1	0.8420.842	6.1×10 ⁹ 6.1×10 ⁹
对照组1Control group 1	0.5850.585	3.7×10 ⁸ 3.7×10 ⁸
对照组2Control group 2	0.6630.663	7.5×10 ⁸ 7.5×10 ⁸
对照组3Control group 3	0.6280.628	6.6×10 ⁸ 6.6×10 ⁸

It can be seen from Table 4 that the bacterial viability and the number of bacteria in the experimental group were significantly higher than those in the control group, indicating that the culture method provided by the present invention can effectively improve the viability and proliferation ability of the magnetotactic bacteria; and at the same time, the bacterial viability of the control groups 2 and 3 The number of bacteria and bacteria were significantly higher than that of the control group 1, indicating that the pre-culture medium and the deep culture medium used in the culture method can effectively improve the viability and proliferation ability of the bacteria.

Experimental example 6

Bio-nanomagnetic beads antibody loading comparison test

The bio-nanomagnetic beads provided in Examples 1 and 4 were used as the experimental groups 1 to 2, and the bio-nano magnetic beads provided in the comparative examples 15 to 16 were used as the control group 1 to 2, and the nano magnetic beads of each group were adsorbed and absorbed. After the water is weighed, the nano magnetic beads are resuspended by adding an appropriate amount of the preservation solution, so that the concentration of the magnetic beads reaches 1 mg/mL. The FITC fluorescently labeled lgG antibody was adjusted to a concentration of 1 mg/mL, and serially diluted with a concentration of 1/10, and the fluorescence intensity of each gradient was calculated by a fluorescence analyzer to prepare a standard curve; another appropriate amount of diluted FITC-labeled antibody was added. 100μL nano magnetic beads, mixed, incubated at 37 °C for 15min, mixed 3~5 times; adsorbed on the nano magnetic beads, absorbed the supernatant, detected the FITC fluorescence intensity of the supernatant, and washed and removed the magnetic beads at the same time In combination with a weak antibody, the fluorescence intensity in the solution was measured after resuspending the magnetic beads, and the antibody loading of each group of nanomagnetic beads was calculated according to a standard curve. The experimental results are shown in Table 5.

Table 5 lgG antibody loading of each group of nanomagnetic beads

组别Group	抗体载量（μg/mg）Antibody loading (μg/mg)
实验组1Experimental group 1	135135
实验组2Experimental group 2	142142
对照组1Control group 1	7979
对照组2Control group 2	8484

As can be seen from Table 5, the antibody loading of the nanomagnetic beads of each group in the experimental group was significantly higher than that of the control group. It is indicated that the present invention can effectively increase the antibody load and improve the reliability of the experimental analysis results by recombinantly expressing the recombinant protein on the bio-nanomagnetic bead membrane protein.

Experimental example 7

High temperature accelerated experiment

The bio-nanomagnetic beads provided in Examples 1 to 4 were used as experimental groups 1 to 4, and 12 sets of parallel experiments were performed for each group of samples. The FITC-labeled lgG antibody was combined with the bio-nano magnetic beads according to the method of Experimental Example 6, and the combination was performed. The bio-nano magnetic beads were placed at 37 ° C, and each sample was taken out on the 1st to 12th day, washed with PBS buffer, resuspended after magnetic adsorption, and the fluorescence intensity of FITC was detected to obtain nano magnetic beads reagent. The decay curve of fluorescence intensity was compared to the reagents normally stored at 4 °C.

As shown in Fig. 7, the bio-nanomagnetic beads from each group in the experimental group retained more than 70% of the original activity after 10 days. It is generally considered that placing at 37 ° C for 1 day is equivalent to placing at 4 ° C for 40 days, and the fluorescence intensity of the bio-nano bead is attenuated by 35% or less. Therefore, it can be considered that the bio-nano magnetic beads provided by the invention can meet the performance standard after being placed at 4 ° C for one year, indicating that it has good stability and can meet the use requirements.

The above-mentioned embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims

A functional biological nanomagnetic bead, characterized in that the membrane protein of the functional bio-nanomagnetic bead is simultaneously fused to express a fluorescent protein and a functional protein.
The functional bio-nanomagnetic bead according to claim 1, wherein the fluorescent protein is any one of EGFP, EBFP, ECFP, EYFP, ERFP, mOrange, mCherry, mStrawberry, mRaspberry, mPlum, and mKate. The EGFP gene sequence is shown in SEQ. ID. No. 1, the EBFP gene sequence is shown in SEQ. ID. No. 2, and the ECFP gene sequence is as shown in SEQ. ID. No. 3. The EYFP gene sequence is shown in SEQ. ID. No. 4, the ERFP gene sequence is shown in SEQ. ID. No. 5, and the mOrange gene sequence is shown in SEQ. ID. No. 6, the mCherry gene. The sequence is as shown in SEQ. ID. No. 7, the mStrawberry gene sequence is shown in SEQ. ID. No. 8, the mRaspberry gene sequence is shown in SEQ. ID. No. 9, and the mPlum gene sequence is as As shown in SEQ. ID. No. 10, the mKate gene sequence is shown in SEQ.
The functional biological nanomagnetic bead according to claim 1, wherein the functional protein is recombinant protein G, recombinant protein A or recombinant streptavidin protein SA, and the genetic sequence of the recombinant protein G is SEQ. As shown in SEQ. ID. No. 13, the gene sequence of the recombinant streptavidin protein A is as shown in SEQ. ID. No. Show.
A method for fluorescently encoding a functional bio-nanomagnetic bead according to any one of claims 1 to 3, comprising the steps of:

S1: Using a magnetotactic bacteria MSR-I to construct a deletion mutant strain of a bacterial magnetic particle membrane protein gene, as a primary recombinant strain, the bacterial magnetic particle membrane protein gene deleted is any two of MamC, MamD and MamF Gene

S2: using any of the deleted bacterial magnetic particle membrane protein gene fusion functional proteins, and introducing into the primary recombinant strain to construct a secondary recombinant strain;

S3: constructing a gene fusion expression vector by using the bacterial magnetic particle membrane protein gene fusion fluorescent protein gene deleted in any one of steps different from step S2; introducing the gene fusion expression vector into the secondary recombinant strain, and Screening to obtain a three-stage recombinant strain having fluorescent coding properties;

S4: The third-stage recombinant strain is cultured to obtain a functional biological nano-magnetic bead capable of simultaneously expressing a fluorescent protein and a functional protein.
The functional bio-nanomagnetic bead fluorescence encoding method according to claim 4, wherein the step S1 comprises the following steps:

S1.1: amplifying a 500 bp homologous DNA fragment flanking the two membrane protein genes, respectively, and constructing two microcarriers by molecular cloning;

S1.2: transferring the two microcarriers into the MSR-I wild type strain simultaneously by electroporation;

S1.3: Screening and identification of the electrotransformed strain to obtain a recombinant strain lacking two kinds, which is a primary recombinant strain.
The functional bio-nanomagnetic bead fluorescence encoding method according to claim 4, wherein the step S2 comprises the following steps:

S2.1: fusing the gene sequence of the functional protein with a deleted sequence of the bacterial magnetic particle membrane protein gene to obtain a new fusion gene fragment;

S2.2: cloning the fusion gene fragment into the expression vector pBRC1 to obtain a functional protein expression plasmid;

S2.3: transferring the functional protein expression plasmid into the primary recombinant strain by means of triple parental ligation or electroporation, and obtaining a recombinant strain expressing the functional protein after verification, that is, a secondary recombinant strain.
The method of claim 4, wherein the method of step S3 is as follows:

S3.1: performing PCR amplification on the fluorescent protein gene sequence;

S3.2: Double digestion with the amplification product and the expression vector pBRC2 by EcoRI/SmaI;

S3.3: the double-digested amplification product is fused with the bacterial magnetic particle membrane protein gene different from the deletion of step S2, and ligated to the expression vector pBRC2 which is also double-digested. Fluorescent protein expression plasmid;

S3.4: transforming the fluorescent protein fusion expression plasmid into the secondary recombinant strain by means of electroporation, and obtaining the tertiary recombinant strain capable of expressing fluorescent protein by screening and verifying;

S3.5: Different fluorescent proteins expressed by the tertiary recombinant strain were analyzed and identified by flow cytometry.
The method of claim 4, wherein the step S4 comprises the following steps:

S4.1: Firstly, using 200~500mL medium for pre-culture, the culture condition is oxygen content 5~10%, N2 content 90~95%, culture time 16 hours, culture temperature 37 °C;

S4.2: transferring the pre-cultured strain to the fermenter for deep culture, the culture condition is 5% oxygen content, hydrogen content 1%, N2 content 94%, culture time 3-4 days, culture temperature 37 ° C;

S4.3: treating the deep culture by homogenizing equipment, pulverizing the cells, adsorbing by a magnetic device, and washing with a phosphate buffer for 2 to 3 times;

S4.4: Gradient treatment with ultrasonic and protease buffer to finally obtain purified functional biological nano magnetic beads.
The method for fluorescently encoding a functional biological nanobead according to any one of claims 5 to 7, wherein the specific method of the electrical conversion is as follows:

Using square wave electric pulse, the electric pulse is 3.1~3.3ms in 1~2 times under the condition of 3100~3200V.
The functional bio-nanomagnetic bead fluorescence encoding method according to claim 8, wherein the medium used in the pre-cultivation comprises the following components:

10~12 parts beef extract, 1~2 parts chitosan, 1~2 parts galactose, 0.5~1 part sodium dihydrogen phosphate, 0.2~0.5 parts phosphatidylserine, 0.05~0.1 parts sodium citrate and 0.5~ 1 part potassium alginate;

The medium used in the deep culture includes the following components:

6~8 parts of Ligustrum lucidum polysaccharide, 8~12 parts of peptone, 1~2 parts of snail polysaccharide, 1~2 parts of lecithin, 0.1~0.2 parts of quercetin, 0.5~1 part of sodium potassium tartrate and 0.05~0.1 parts of lemon Sodium.