CN112921030A

CN112921030A - Preparation method of high-yield nucleic acid fragment

Info

Publication number: CN112921030A
Application number: CN202110396644.7A
Authority: CN
Inventors: 张永正; 马诗敏; 张征立; 李洋; 康涛
Original assignee: Jiangsu Yao Hai Biopharmaceutical Co ltd
Current assignee: Jiangsu Yao Hai Biopharmaceutical Co ltd
Priority date: 2021-04-13
Filing date: 2021-04-13
Publication date: 2021-06-08

Abstract

The invention is applicable to the technical field of biology, and provides a preparation method of a high-yield nucleic acid fragment, which comprises the following steps: respectively introducing recognition sites of the same restriction enzyme into 5 'and 3' ends of a target gene sequence so as to obtain a target gene fragment through single enzyme digestion and ensure the uniformity of the fragment; the target gene fragment and the vector framework are connected into a recombinant plasmid containing the target gene fragment of at least 2Copy by enzyme digestion and connection. Amplifying and collecting recombinant plasmids, and carrying out enzyme digestion on the recombinant plasmids by using single restriction enzyme to obtain enzyme digestion solution; and separating and purifying the enzyme digestion solution by using AKTA through anion exchange resin to obtain a target gene fragment. The method realizes high yield of target gene fragments in a single batch, reduces the scale and batch amount of fermentation and purification, shortens the production period, improves the utilization rate of equipment, and increases the economic benefit.

Description

Preparation method of high-yield nucleic acid fragment

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method of a high-yield nucleic acid fragment.

Background

The preparation of the nucleic acid fragment is mainly obtained by synthesis, a PCR amplification method and digestion after plasmid amplification, wherein the PCR method is difficult to ensure batch uniformity due to the existence of low-probability mutation in the processes of in vitro polymerase identification, addition and cutting, the nucleic acid fragment obtained by digestion from the plasmid can effectively ensure the stability of a gene sequence due to a complex replication mechanism in a cell, and the restriction endonuclease is specifically cut to ensure the batch consistency of the target nucleic acid fragment. Therefore, the plasmid digestion method is usually preferred to obtain a large amount of target nucleic acid fragments with uniform quality.

In the preparation process of the nucleic acid fragment, a target nucleic acid fragment is obtained through PCR amplification or complete sequence synthesis, the target nucleic acid fragment is inserted into a plasmid vector to obtain a recombinant plasmid, and a large number of target nucleic acid fragments are finally obtained through amplification and enzyme digestion of the recombinant plasmid. Generally, a recombinant plasmid only contains one Copy target nucleic acid fragment, when the demand of the target nucleic acid fragment is low, the target nucleic acid fragment with low yield can meet the demand, but when the demand is high, the problem of low yield of the target nucleic acid fragment becomes abnormal and prominent, the demand of materials is increased, the production cycle is prolonged, and the economic benefit is directly low. Therefore, how to increase the content of the target nucleic acid fragment in a unit plasmid becomes a great problem to be solved urgently for increasing economic benefit.

Disclosure of Invention

It is an object of the embodiments of the present invention to provide a method for preparing a highly productive nucleic acid fragment, which aims to solve the problems set forth in the background art.

The embodiment of the present invention is achieved by a method for preparing a high-yielding nucleic acid fragment, which comprises the steps of:

respectively introducing recognition sites of the same restriction enzyme at the 5 'end and the 3' end of a target gene sequence so as to obtain a target gene fragment by single enzyme digestion and ensure the uniformity of the fragment;

the target gene fragment and the vector framework are connected into a recombinant plasmid containing the target gene fragment of at least 2Copy by enzyme digestion and connection.

Amplifying and collecting recombinant plasmids, and carrying out enzyme digestion on the recombinant plasmids by using single restriction enzyme to obtain enzyme digestion solution;

separating and purifying the enzyme digestion solution by using AKTA through anion exchange resin, and collecting a target gene fragment.

As another preferable embodiment of the present invention, in the step, the recognition sites of the restriction enzymes introduced are the same enzyme.

As another preferable scheme of the embodiment of the invention, in the step, the recombinant plasmid contains 2-10 Copy target gene segments.

As another preferable scheme of the embodiment of the invention, in the step, the recombinant plasmid contains 2-3 Copy target gene segments.

As another preferred scheme of the embodiment of the present invention, in the step, the target gene fragment is obtained by obtaining an enzyme-cleaved product containing the target gene fragment by direct enzyme cleavage or a method combining PCR and enzyme cleavage, and performing electrophoretic separation, recovery, and purification to obtain the target gene fragment.

As another preferred scheme of the embodiment of the present invention, in the step, the vector skeleton is a dephosphorylated vector skeleton fragment, which is obtained by directly performing enzyme digestion or by combining PCR and enzyme digestion to obtain an enzyme digestion product of the vector skeleton fragment containing the ORI region and the antibiotic resistance gene region, and the enzyme digestion product is subjected to electrophoretic separation, recovery and purification to obtain the vector skeleton fragment; and then, carrying out dephosphorylation treatment on the carrier framework fragment, and then purifying and recovering to obtain the dephosphorylated carrier framework fragment.

As another preferred embodiment of the present invention, the antibiotic resistance gene is kanamycin resistance gene to avoid antibiotic residues and diffusion of the resistance gene to the environment.

According to the preparation method of the high-yield nucleic acid fragment provided by the embodiment of the invention, the content of the target gene fragment in the plasmid is increased from the plasmid level according to the concept of 'quality source design', so that the content of the plasmid in unit fermentation liquor is increased, and the yield of the target gene fragment in a single batch is increased.

The target gene can be inserted in different enzyme cutting sites, can be inserted after the same enzyme cutting site is connected in series, can be inserted after the same tailase is cut and connected in series, and can also be in a mixed form.

According to the design, the target gene fragment and the vector skeleton fragment can be respectively obtained by direct enzyme digestion or a method combining PCR and enzyme digestion.

According to the design, the target gene segment or the self-connection segment thereof is connected with the dephosphorylation vector skeleton segment, transformed and subjected to resistance screening to obtain the recombinant plasmid containing a plurality of Copy target gene segments.

The target gene self-connection fragment is obtained by reacting a target gene fragment with ligase, spontaneously connecting the target gene fragment and the ligase, performing agarose gel electrophoresis, and then performing gel cutting, recovery and purification treatment.

In addition, the recombinant plasmid is amplified and collected, and a plurality of target gene segments can be cut from the recombinant plasmid by utilizing single restriction enzyme for enzyme digestion. And then, carrying out sample loading analysis on the enzyme digestion solution by using AKTA, separating and purifying by using anion exchange resin, collecting a target peak and a carrier peak, analyzing the peak area, and detecting and determining the content of a target gene fragment and the content of a carrier in the sample. The method has simple and convenient enzyme digestion operation, the yield of the target gene fragment in a single batch of plasmid sample is high, the influence of the vector fragment on the separation of the target gene fragment is reduced due to the low content of the vector fragment in the sample, the content of the target gene fragment in the sample is high, and the separation and the purity improvement are easy.

The present invention provides an example of the method for preparing nucleic acid fragments according to the above: the embodiment can prepare the recombinant plasmid containing the target gene fragment of 2copy or more, does not influence the copy number of the whole plasmid, improves the plasmid yield obtained by single batch of thalli, increases the yield of the target gene fragment obtained in single batch of purification work, realizes high yield of the target gene fragment in single batch of plasmid samples, shortens the production period, improves the utilization rate of equipment and increases economic benefits.

Drawings

FIG. 1 is a restriction map of plasmid pUC 57-Kan-Yh; wherein, M: DNA Marker; 1: KpnI digestion of plasmid pUC 57-Kan-Yh.

FIG. 2 is a recovered Yh self-assembly fragment electrophoresis detection map; wherein, M: DNA Marker; 1: yh self-ligating fragment.

FIG. 3 is a diagram showing the result of identification of a recombinant plasmid; wherein, M: DNA Marker; in A, 1-5 are plasmid electrophoresis detection; b, performing enzyme digestion electrophoresis detection on 1-5 plasmids NdeI; and C, 1-5 is a plasmid KpnI enzyme digestion electrophoresis detection.

FIG. 4 is a graph showing the results of analysis of plasmid yields and target gene fragment yields of different plasmid strains, wherein 1 to 5 are respectively 2Yh01, 2Yh02, 2Yh03, 2Yh04 and 2Yh 05.

FIG. 5 is a graph showing the results of comparative analyses of the plasmid yield and the yield of a target gene fragment, in which 2Yh01 is a recombinant plasmid.

FIG. 6 is a structural diagram of a recombinant plasmid.

FIG. 7 is a diagram of enzymatic cleavage electrophoresis detection prior to purification; wherein, 1 is a raw plasmid sample; 2 is a recombinant plasmid sample; m is DNA Marker.

FIG. 8 is an electrophoretic detection chart of the recovered target gene fragment after purification; wherein M is a DNA Marker; 1 is a recombinant plasmid sample recovery fragment; 2 is the original plasmid-like recovered fragment.

FIG. 9 is a chromatogram and chromatogram integration result chart of recombinant plasmid-like preparation fragments. Wherein, fig. 9 (b) is a partial enlarged view of the chromatogram in fig. 9 (a).

FIG. 10 is a chromatogram and chromatogram integration result chart of the crude plasmid sample preparation fragment. Wherein, fig. 10 (b) is a partial enlarged view of the chromatogram in fig. 10 (a).

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

This example provides a method of practicing high-yielding nucleic acid fragment preparation, comprising the steps of:

EXAMPLE 1 construction of recombinant plasmid

S1, taking a strain of pUC57-Kan-Yh plasmid containing target genes, inoculating the strain into LB liquid culture medium containing 50 mu g/mL Kan, carrying out overnight shaking culture at 37 ℃ and 220rpm, taking an overnight culture, and carrying out plasmid extraction by using a plasmid small quantity extraction kit to obtain the pUC57-Kan-Yh target plasmid.

S2, taking 10 mu g of pUC57-Kan-Yh target plasmid, carrying out enzyme digestion by using restriction enzyme KpnI, carrying out enzyme digestion for 40min at 37 ℃, carrying out electrophoresis separation on reaction liquid by using 1% agarose gel, cutting gel, recovering a target fragment band of about 3759bp and a carrier fragment of 2579bp, and purifying by using a DNA gel recovery kit to obtain a Yh target gene fragment and a carrier skeleton fragment, wherein the DNA gel recovery kit is shown in figure 1.

S3, adding Calf Intestinal Alkaline Phosphatase (CIAP) into the recovered vector skeleton fragment solution for dephosphorylation treatment, reacting at 50 ℃ for 60min, and then directly using a gel recovery kit to purify and recover the dephosphorylated vector skeleton fragment to obtain the pUC57-Kan dephosphorylated vector skeleton fragment.

S4, taking the recovered Yh target gene fragment, carrying out self-Ligation reaction on the Yh target gene fragment by using a DNA Ligation Mix, carrying out Ligation reaction for 5min at 16 ℃, carrying out electrophoresis separation by using 1% agarose gel, cutting gel, recovering a target fragment band of about 7518bp, and purifying by using a DNA gel recovery kit to obtain the Yh target gene self-Ligation fragment, wherein the fragment is shown in figure 2.

S5, taking the dephosphorylation carrier skeleton fragment, the Yh target gene self-connection fragment and the DNA Ligation Mix to form 8.6 mu L of connection liquid, wherein the connection system is shown in the following table 1:

TABLE 1

Components	Concentration (ng/. mu.L)	Volume (μ L)
			Dephosphorylated vector backbone fragments	23.5	0.3
Yh target gene self-linking fragment	5.3	4
			LigationMix	-	4.3
General System	-	8.6

And (3) performing a ligation reaction on the ligation system at 16 ℃ for 20min, and then placing the ligation system in an ice bath to obtain a ligation solution for later use.

S6, gently mixing the connecting liquid with Escherichia coli competent cell Trans10, placing the mixture in an ice bath for 30min, thermally shocking 90Sec at 42 ℃, rapidly placing the mixture in the ice bath for 2min, adding 200 mu L of nonreactive LB culture medium, incubating and recovering for 1h at 37 ℃, and then coating a Kan resistant LB plate containing 50 mu g/mL; then, the coated resistant plate is taken out, kept stand for 5min, placed upside down and cultured in a constant temperature incubator at 37 ℃ until obvious colonies appear (the culture time is not more than 20 h). And (4) positive clones growing on the resistant plate are the target strains suspected to contain the recombinant plasmids.

S7, taking the screened target strain, inoculating the target strain into a Kan resistant LB liquid culture medium containing 50 mu g/mL, and carrying out shake culture at 37 ℃ and 220rpm overnight to obtain a plasmid strain culture; taking a plasmid strain culture, and extracting plasmids by using a plasmid small-quantity extraction kit to obtain a plasmid solution of the plasmid strain culture; then, 1 μ L of plasmid solution is separated and detected by 1% agarose gel electrophoresis, and the size of the detected band is consistent with the theoretical value (about 10097bp), thus obtaining the target recombinant plasmid.

In addition, the enzyme digestion identification method of the recombinant plasmid is as follows:

taking 1 mu g of extracted recombinant plasmid, separately digesting with restriction enzyme NdeI or KpnI, performing digestion reaction at 37 ℃ for 40min, separating and detecting the digestion product by 1% agarose gel electrophoresis, wherein the size of a band obtained after digestion with the restriction enzyme NdeI is a single band and is consistent with theoretical expectation (about 10097bp), the size of a band obtained after digestion with the restriction enzyme KpnI is a double band and is consistent with the theoretical expectation (about 3759bp and 2579bp), and the obtained plasmid is the target recombinant plasmid containing 2copy Yh fragment, as shown in FIG. 6. The detection results show that the recombinant plasmid containing the double-copy Yh fragment is successfully constructed and is named as pUC 57-Kan-Yh-Yh. The obtained plasmid bacteria were named 2Yh01, 2Yh02, 2Yh03, 2Yh04 and 2Yh05, respectively.

Example 2 screening of high-producing plasmid strains and analysis of the yields of the desired gene fragments of recombinant plasmids:

screening of high-yield plasmid strains

5 newly-prepared plasmid strains and the original plasmid strain which are correctly identified are taken and inoculated into Kan resistant LB liquid culture medium containing 50 mu g/mL respectively, and are cultured under shaking at 37 ℃ and 220rpm overnight. OD of each bacterial liquid was measured at 600nm in a spectrophotometer₆₀₀The value is obtained. Adjusting each bacterial liquid to proper OD with sterile water₆₀₀The appropriate volume of the bacterial liquid was taken, the plasmid was extracted with a plasmid mini-extraction kit, and the same volume (100. mu.L) was used for elution. Then, the concentration of each plasmid solution is measured by an ultramicro spectrophotometer, and the content of the plasmids and the content of the target gene fragments in each sample are calculated. According to the content of the target gene fragment, the plasmid with higher yield of the target gene fragment and the corresponding plasmid strain thereof are screened out.

After amplification culture under the same conditions, plasmid extraction is carried out on the same bacterial quantity, and the plasmid content in OD bacterial liquid of 5 plasmid strains 2Yh 01-2 Yh05 is respectively 4.80 mu g, 2.41 mu g, 4.57 mu g, 4.69 mu g and 3.62 mu g, and the plasmid content in OD bacterial liquid of the original plasmid strain is 3.47 mu g. Relative quantification was performed and plotted using the plasmid and theoretical target gene content of the original plasmid as references, see FIG. 4. Through analysis, the plasmid contents of the strains 2Yh01, 2Yh03 and 2Yh04 are all higher than those of original plasmid bacteria, and are averagely improved by about 35 percent; the original plasmid size is 6338bp, the newly constructed plasmid size is 10097bp, and after conversion, the molar weight is slightly lower than that of the original plasmid, which indicates that the plasmid copy number in each strain is not obviously different from the original plasmid; because the original plasmid contains 1co py target gene fragment and the new plasmid contains 2copy target gene fragment, the new plasmid has higher theoretical target gene fragment yield after conversion, reaches 2 times of the target gene fragment yield of the original plasmid, and is consistent with theoretical expectation. As a result, the recombinant plasmid strain 2Yh01 was preferably used for subsequent studies.

Second, analysis of yield of target gene fragment of recombinant plasmid

Selecting original plasmid strains and recombinant plasmid strains, respectively carrying out shake flask amplification culture, carrying out amplification culture under the same conditions, taking equivalent bacterial quantity to carry out plasmid extraction, analyzing the plasmid content, and measuring the plasmid content in OD bacterial liquid to be 1.85 mu g and 3.74 mu g respectively. Relative quantitative analysis was performed and plotted using the plasmid and theoretical target gene content of the original plasmid as references, see FIG. 5. The plasmid content of the recombinant plasmid strain is higher than that of the original plasmid strain through analysis, and is improved by about 1 time; according to the size of the plasmid, after conversion, the molar weight is slightly higher than that of the original plasmid, which shows that the copy number of the plasmid in the recombinant plasmid strain is not obviously different from that of the original plasmid; according to the theoretical content of the target gene fragment in the plasmid, the theoretical target gene fragment yield of the recombinant plasmid is higher after conversion, reaches 2 times of the target gene fragment yield of the original plasmid, and is consistent with theoretical expectation.

EXAMPLE 3 isolation and purification analysis of the Gene fragment of interest

(1) Plasmid amplification and extraction

The recombinant plasmid strain and the original plasmid strain are respectively taken and inoculated in Kan resistant LB liquid culture medium containing 50 mug/mL, and are subjected to shaking culture at 37 ℃ and 220rpm overnight. Respectively taking the bacterial liquid, centrifuging at 8000 Xg for 2min, collecting thallus, and respectively extracting plasmids.

(2) Digestion by enzyme

The recombinant plasmid and the original plasmid of 1mg are respectively cut by restriction enzyme KpnI, and electrophoresis detection is carried out until the enzyme cutting is complete, as shown in figure 7.

(3) Purification of target Gene fragments

Taking a certain amount of enzyme digestion solution, and carrying out sample loading analysis by using AKTA. Separating and purifying by a chromatographic column NanoQ-15L, and respectively collecting a target peak and a carrier peak. Analyzing peak area and detecting and determining the content of target gene fragments and the content of carriers in the two samples. The method comprises the following specific steps:

equilibration of the column: a NanoQ-15L column (21.2X 150mM) was equilibrated with solution A (10mM Tris-HCl, 1mM EDTA, 0.4M NaCl, pH8.0) at a flow rate of 4ml/min for 3 column volumes. At the end of the equilibration, the UV detection wavelength A260 was adjusted to zero and ready for loading.

Loading: the cut solution containing 0.92mg of plasmid was sampled at a flow rate of 4 ml/min.

Washing 1: and (3) washing the loaded chromatographic column by using the solution A at the flow rate of 4ml/min, and washing the column by 3 column volumes to reduce the ultraviolet detection value under the wavelength of 260nm to be stable.

And (3) washing 2: the solution A is used as a base solution and mixed with a solution B (10mM Tris-HCL, 1mM EDTA, 1M NaCl, pH8.0) to prepare a mixed solution with a linear proportion of 0-40% B, and the mixed solution is subjected to linear elution and washing by using 2 column volumes of the mixed solution at a flow rate of 4 ml/min.

And (3) elution: preparing the solution A and the solution B into a linear proportion mixed solution of 40-65% of solution B, performing linear elution by using 15 column volume mixed solutions, collecting an ultraviolet detection value larger than 5mAu absorption peak under the wavelength of 260nm at the flow rate of 4ml/min, and respectively collecting a target fragment peak and a carrier fragment peak.

And (3) detection: the concentration of the target gene fragment is determined by ultraviolet method of Nano ultramicro spectrophotometer, and the size of the target gene fragment is detected by agarose gel electrophoresis, as shown in figure 8.

From the electrophoresis results of FIG. 8, it was revealed that the size of the recovered fragment was consistent with the theoretical expectation (about 3759bp), and it was the target gene fragment; and the single band and no miscellaneous band indicate that the recovered fragment solution is pure target gene fragment solution, and the ultraviolet detection result is the actual content thereof.

In addition, comparative analysis of the purification effect of the objective gene fragment in the above two plasmid samples is shown in Table 2.

TABLE 2

Under the condition of the same plasmid amount from the table 2, the amount of the target gene fragment recovered from the recombinant plasmid enzyme digestion sample is higher than that of the original plasmid sample, and the amount of the recovered vector fragment is obviously lower than that of the original plasmid sample; the proportion of the recovered target gene fragment in the total amount of the recovered DNA fragment is 15.1% higher than that of the original plasmid, and is consistent with the theoretical increase (15.2%) of the content of the target gene fragment in unit plasmid amount, which shows that under a certain total DNA recovery rate of unit plasmid amount, the higher the initial content of the target gene fragment is, the higher the amount of the recovered target gene fragment is. In the experiment, the yield of the target gene fragment in the batch of recombinant plasmid samples is only improved by 3.8 percent compared with the original plasmid samples, and is greatly different from the theoretical yield increase by 25.6 percent, because the total amount of the recovered DNA after the purification of the recombinant plasmid samples is lower, the recovery rate is lower by 14.3 percent compared with the original plasmid samples, and the yield increase of the target gene fragment is limited. Therefore, under a certain total DNA recovery rate, the recombinant plasmid can obviously improve the utilization efficiency of the purified filler medium and the yield of target gene fragment products in a single batch, shorten the production period and improve the production efficiency.

(4) Purity detection of target gene fragment

And (4) taking the collected target gene fragment solution, respectively detecting by using HPLC, and analyzing the purity of the sample. Using a column TSKgel DNA-NPR (4.6 mM. times.7.5 cm), 100. mu.L of the sample was injected at 25 ℃ and the absorbance peak was detected at 260nm by linear gradient elution using solution A (20mM Tris-HCl, pH 8.8) and solution B (20mM Tris-HCl, 1M NaCl, pH 8.8) at a flow rate of 0.5mL/min as shown in FIG. 9 and FIG. 10.

According to the integration result of a chromatographic spectrogram, the purity of the recombinant plasmid-like preparation fragment (S1 sample, figure 9) is 97.75%, the purity of the original plasmid sample preparation fragment (D1 sample, figure 10) is 98.00%, the purity meets the requirement, and the purity difference is not obvious, so that the purity of subsequent purification is not influenced while the yield of the target gene fragment is improved by the recombinant plasmid prepared by the method.

In summary, the embodiment of the present invention provides a new recombinant plasmid constructed according to the preparation method of the present invention, which contains 2copy target gene fragments, and does not affect the whole plasmid copy number and plasmid yield, so as to improve and increase the yield of target gene fragments obtained from a single batch, achieve the purpose of high yield of target gene fragments, shorten the production cycle, improve the equipment utilization rate, and increase the economic benefits.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for preparing a highly productive nucleic acid fragment, comprising the steps of:

respectively introducing recognition sites of the same restriction enzyme into 5 'and 3' ends of a target gene sequence so as to obtain a target gene fragment through single enzyme digestion and ensure the uniformity of the fragment;

connecting the target gene segment and a vector framework by means of enzyme digestion and connection to form a recombinant plasmid containing at least 2Copy of the target gene segment;

separating and purifying the enzyme digestion solution by using AKTA through anion exchange resin to obtain a target gene fragment, and detecting the purity of the target gene fragment by using HPLC.

2. The method for preparing a highly productive nucleic acid fragment as claimed in claim 1, wherein the recognition sites of the restriction enzymes introduced in said step are the same enzyme.

3. The method for preparing a high-yield nucleic acid fragment as claimed in claim 1, wherein in the step, the recombinant plasmid contains 2-10 Copy target gene fragments.

4. The method for preparing a high-yield nucleic acid fragment as claimed in claim 3, wherein the recombinant plasmid contains 2-3 Copy target gene fragments.

5. The method for preparing the high-yield nucleic acid fragment according to claim 1, wherein in the step, the target gene fragment is obtained by directly performing enzyme digestion or by combining PCR with enzyme digestion to obtain an enzyme digestion product containing the target gene fragment, and the target gene fragment is obtained by electrophoretic separation, recovery and purification.

6. The method for preparing the high-yield nucleic acid fragment according to claim 1, wherein in the step, the carrier skeleton is a dephosphorylated carrier skeleton fragment, and the enzyme digestion product of the carrier skeleton fragment containing the ORI region and the antibiotic resistance gene region is obtained by direct enzyme digestion or a method combining PCR and enzyme digestion, and the carrier skeleton fragment is obtained by electrophoretic separation, recovery and purification treatment; and then, carrying out dephosphorylation treatment on the carrier framework fragment, and then purifying and recovering to obtain the dephosphorylated carrier framework fragment.

7. The method for preparing a nucleic acid fragment with high yield as claimed in claim 1, wherein the step of obtaining the DNA fragment containing 2copy target gene is carried out by self-ligation, isolation and recovery of the target gene fragment, and the DNA fragment is ligated with dephosphorylated vector backbone fragment and transformed to obtain recombinant plasmid.

8. The method for preparing a high-yield nucleic acid fragment as claimed in claim 1, wherein in the step, the recombinant plasmid is subjected to single enzyme digestion, and is separated and purified by AKTA anion exchange resin to obtain the target gene fragment, and the purity of the target gene fragment is detected by HPLC.

9. A recombinant plasmid containing at least two copies of a target gene fragment prepared by the method according to any one of claims 1 to 8.