阅读说明：本技术 一种卤化酶重组表达载体制备方法及其应用 (Preparation method and application of halogenase recombinant expression vector ) 是由 王苗 李昱良 刘建国 王倩 任群利 胡欢 李小兰 于 2021-01-07 设计创作，主要内容包括：本发明以链霉菌Streptomyces sp.FJS31-2基因组为模板,特异引物扩增卤化酶片段,TA克隆,T载体通用引物进行菌液PCR鉴定,abx H/T质粒BglⅡ和HindⅢ限制性内切酶切鉴定并胶回收目的片段,连接相同酶切并去磷酸化处理的质粒载体,连接产物室温过夜连接,转化克隆菌株JM109感受态细胞。进行特异引物菌液PCR鉴定,扩增出目的条带的阳性克隆提质粒限制性内切酶酶切鉴定,切出载体片段和插入的目的条带。测序验证的阳性菌转化表达菌株BL21(DE3)Rosetta感受态细胞,特异引物菌液PCR鉴定。在不同温度下进行IPTG诱导表达,蛋白样品处理,SDS-PAGE蛋白质电泳分析,可见很明显的诱导蛋白条带,卤化酶大小为65KD。(The invention uses streptomycete Streptomyces Taking an sp.FJS31-2 genome as a template, amplifying a halogenase fragment by using a specific primer, cloning TA, carrying out bacteria liquid PCR identification by using a T carrier general primer, carrying out restriction enzyme digestion identification on abx H/T plasmids Bgl II and Hind III, recovering a target fragment by glue, connecting plasmid carriers which are subjected to the same enzyme digestion and dephosphorylation treatment, connecting a connecting product at room temperature overnight, and transforming a clone strain JM109 competent cell. Carrying out PCR identification on specific primer bacterial liquid, amplifying positive clone of a target band, carrying out restriction enzyme digestion identification on plasmid, cutting out a vector fragment and an inserted target band. And (3) transforming expression strain BL21(DE3) Rosetta competent cells by positive bacteria verified by sequencing, and carrying out PCR identification on specific primer bacteria liquid.IPTG induced expression, protein sample treatment and SDS-PAGE protein electrophoresis analysis are carried out at different temperatures, a very obvious induced protein band can be seen, and the size of the halogenase is 65 KD.)

1. A preparation method of a halogenase recombinant expression vector is characterized in that: the sequence of the halogenase is shown as SEQ ID NO.1, and the halogenase comprises the following steps:

1) according to the Streptomyces spStreptomyces31-2 genome sequencing result to design complete coding region amplification primer of coding gene, and restriction endonuclease is added to upstream and downstream primers of Halo coding gene respectivelyBgl II and HindIII recognition sites, and the sequence of a primer HaloER is as follows: 5'-CCCAAGCTTCGGCATGGCGGTCTTCATGT-3', the sequence of primer HaloEF is: 5'-GGAAGATCTATGGACACGACAGACACAGGCG-3', respectively;

2) HaloEF/ER halogenase specific primers andStreptomycessp, FJS31-2 genome DNA as template amplification, PCR product purification, TA cloning, universal primer M13F/M13RCarrying out PCR verification, sequencing verification of positive cloning, carrying out double enzyme digestion on the strain Halo-T by using restriction enzymes Bgl II and Hind III, connecting double enzyme digestion fragments with an expression vector pET-32a which is subjected to corresponding enzyme digestion and dephosphorylation treatment, carrying out room temperature reaction for 6h, transforming JM109 competent cells, carrying out PCR identification on specific primer bacterium liquid and plasmid enzyme digestion identification, and carrying out sequencing identification to obtain a prokaryotic expression plasmid vector pET-Halo; finally obtaining positive clone bacteria plasmid transformed BL21(DE3) Rosetta competent cells, carrying out PCR identification on specific primer bacteria liquid, and determining positive clone bacteria;

3) taking 100ng of plasmid of positive clone bacterium pET-halo and plasmid pET32a, transforming escherichia coli BL21(DE3) Rosetta competent cells, carrying out overnight culture on transformants, and carrying out PCR (polymerase chain reaction) identification on T7 promoter liquid to obtain genetically engineered bacteria pET-halo BL21 and pET32aBL21 of recombinant plasmids;

4) respectively inoculating 50 mu L of the 2 kinds of genetic engineering bacteria into 5mL of LB liquid culture medium with concentration of 100 ng/mu L ampicillin at 220r/min, respectively inoculating 100mL of LB liquid culture medium with concentration of 100 ng/mu L ampicillin at 220r/min after culturing at 37 ℃ for 3h, respectively, subpackaging 5mL of the culture into 50mL of sterile centrifuge tubes when culturing at 37 ℃ till OD600=0.3, adding IPTG with final concentration of 1.0mmol/L, and placing in a constant temperature shaking table for inducing for 6 h; centrifuging 1mL of the induced culture for 5min at 4000r/min, adding 100 mu L of 5 xSDS-PAGE electrophoresis sample buffer solution into the thallus precipitate, placing the thallus precipitate in a 99 ℃ metal bath for cracking for 10min at 12000r/min, and centrifuging at room temperature for 10min to obtain a supernatant which is a thallus total protein lysate;

5) and respectively taking 10 mu L of total protein lysate for sampling, performing 10% SDS-PAGE gel electrophoresis, dyeing for 6h by Coomassie brilliant blue R250, and decoloring for 6h by using methanol-glacial acetic acid decolorant, wherein the result shows that the target halogenase protein is obtained.

Technical Field

The invention relates to the field of genetic engineering, in particular to a preparation method and application of a halogenase recombinant expression vector.

Background

Halides are a class of organic compounds containing the halogen atom fluorine, chlorine, bromine or iodine. Most halides have important biological activity, and many clinically used antibiotics and antitumor drugs are halides derived from microorganisms, such as chloramphenicol and vancomycin with antibacterial activity, and butterfly mycin, C-1027 with antitumor activity. 3 of the 4 new antibiotics approved for marketing in 2014 were halides, dalvanine, Oritavanci, and Sivextro, respectively.

The halogen atom in the halide has important influence on the physiological activity of the halide, so that the related activity of the compound can be effectively improved, the half-life period can be prolonged, and the bioavailability can be increased. Traditional halide mining relies primarily on a pure culture method that is found to be highly reproducible and not directional.

The use of microorganisms is also an important source of halide discovery.

Disclosure of Invention

The invention aims to provide a halogenase heterologous expression vector and construction thereof by utilizing microbial technology on the basis of the background technology.

In order to achieve the purpose, the technical scheme is as follows:

the sequence of the halogenase is shown as SEQ ID NO. 1;

1) designing complete coding region amplification primers of a coding gene according to a sequencing result of a Streptomyces 31-2 genome, adding restriction enzyme BglII and HindIII recognition sites into upstream and downstream primers of a Halo coding gene respectively, wherein the sequence of the primer HaloER is as follows: 5'-CCCAAGCTTCGGCATGGCGGTCTTCATGT-3', the sequence of primer HaloEF is: 5'-GGAAGATCTATGGACACGACAGACACAGGCG-3', respectively;

2) amplifying a specific primer of HaloEF/ER halogenase by taking Streptomyces sp.FJS31-2 genome DNA as a template, purifying a PCR product, cloning TA, carrying out PCR verification by using a general primer M13F/M13R, carrying out sequencing verification on positive cloning, carrying out double enzyme digestion on a strain Halo-T by using restriction enzymes Bgl II and Hind III, connecting double enzyme digestion fragments with an expression vector pET-32a which is subjected to corresponding enzyme digestion and dephosphorylation treatment, reacting at room temperature for 6h, transforming JM109 competent cells, carrying out PCR identification on specific primer bacterium liquid and plasmid enzyme digestion identification, and carrying out sequencing identification to obtain a prokaryotic expression plasmid vector pET-Halo; finally obtaining positive clone bacteria plasmid transformed BL21(DE3) Rosetta competent cells, carrying out PCR identification on specific primer bacteria liquid, and determining positive clone bacteria;

3) taking 100ng of plasmid of positive clone bacterium pET-halo and plasmid pET32a, transforming escherichia coli BL21(DE3) Rosetta competent cells, carrying out overnight culture on transformants, and carrying out PCR (polymerase chain reaction) identification on T7 promoter liquid to obtain genetically engineered bacteria pET-halo BL21 and pET32aBL21 of recombinant plasmids;

4) respectively inoculating 50 mu L of the 2 kinds of genetic engineering bacteria into 5mL of LB liquid culture medium with concentration of 100 ng/mu L ampicillin for 220r/min, respectively inoculating 100mL of LB liquid culture medium with concentration of 100 ng/mu L ampicillin for 220r/min after culturing for 3h at 37 ℃, respectively subpackaging 5mL of the culture into 50mL of sterile centrifuge tubes when culturing at 37 ℃ till OD600 is 0.3, adding IPTG with final concentration of 1.0mmol/L, and placing in a constant temperature shaker for inducing for 6 h; centrifuging 1mL of the induced culture for 5min at 4000r/min, adding 100 mu L of 5 xSDS-PAGE electrophoresis sample buffer solution into the thallus precipitate, placing the thallus precipitate in a 99 ℃ metal bath for cracking for 10min at 12000r/min, and centrifuging at room temperature for 10min to obtain a supernatant which is a thallus total protein lysate;

5) and respectively taking 10 mu L of total protein lysate for sampling, performing 10% SDS-PAGE gel electrophoresis, dyeing for 6h by Coomassie brilliant blue R250, and decoloring for 6h by using methanol-glacial acetic acid decolorant, wherein the result shows that the target halogenase protein is obtained.

Research shows that the biosynthetic Gene cluster (Gene cluster) of the halide exists in the genome of the microorganism, and the halogenase Gene in the Gene cluster is responsible for the halogenation modification of the compound, plays an important role in the biosynthesis pathway of antibiotics under the catalysis of the halogenase, and can greatly improve the bacteriostatic activity of metabolites. The halogenase is an important late-modifying enzyme capable of conferring activity on secondary metabolites, has substrate specificity, and is likely to catalyze the synthesis of new halogenides. The halogenase gene is used as a probe, and a natural biosynthesis gene cluster coupled with the halogenase gene can be found, thereby providing a basis for the discovery of halogenide.

Streptomyces sp.FJS31-2 genome is taken as a template, a target fragment of a halogenase abx H is amplified by a specific primer, TA cloning is carried out, a general primer M13F/M13R primer of a T carrier is used for carrying out bacteria liquid PCR identification, restriction enzyme digestion identification and gel recovery are carried out on abx H/T plasmids Bgl II and Hind III to obtain a 1300bp target fragment, the plasmid carrier pET32a which is subjected to the same enzyme digestion and dephosphorylation treatment is connected, and a connecting product is connected overnight at room temperature to transform a competent cell of a cloned JM 109. Is named as pET-abxH-J. The PCR identification of specific primer bacterial liquid is carried out by pET-abxH-J, the corresponding target strip 1300bp (figure 1) can be amplified, the restriction enzyme digestion identification of plasmid can be carried out by the positive clone of the amplified target strip, and the 5900bp of the carrier fragment and the 1300bp of the inserted target strip can be cut out. Positive bacteria transformation expression strain BL21(DE3) Rosetta competent cells verified by sequencing, specific primer bacteria liquid PCR identification (figure 2), respectively named pET-abxH R, IPTG induced expression at different temperatures (18 ℃, 28 ℃ and 37 ℃), protein sample treatment and SDS-PAGE protein electrophoresis analysis show that an induced protein band is obvious (figure 3), and the size of the halogenase is 65 KD.

Drawings

FIG. 1 shows the identification of primers specific to competent cells of JM109 transformed with pET-abxH-J ligation according to the present application.

FIG. 2 shows the identification of primers specific to Rosetta competent cells transformed with pET-abxH J ligation BL21(DE3) according to the present application.

FIG. 3 shows the electrophoretic analysis of the protein expressed by pET-abxH R induction in the present application.

Detailed Description

The technical solution of the present invention will be clearly and completely described below with reference to the specific embodiments of the present invention. Referring to the drawings, like numbers indicate like or similar elements throughout the views. The described embodiments are only some, not all embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The sequence of the halogenase is shown in SEQ ID NO. 1.

FIG. 1 shows the identification of primers specific to competent cells of JM109 transformed with pET-abxH-J ligation according to the present application. Wherein 1/2/3/7/8/16/20 can amplify 1300bp of Halo target gene, which is positive clone.

FIG. 2 shows the identification of primers specific to Rosetta competent cells transformed with pET-abxH J ligation BL21(DE3) according to the present application. All of them, except 8, were positive clones.

FIG. 3 shows the electrophoretic analysis of the protein expressed by pET-abxH R induction in the present application. BSA is bovine serum albumin, has molecular weight of 66.430kDa, has a size which is not greatly different from that of the identified halogenase gene of 65kDa, and is taken as marker. 1 is pET-abxH R strain without IPTG addition, and 2, 3 and 4 are pET-abxH R strains cultured with IPTG addition at 18 deg.C, 28 deg.C and 37 deg.C, respectively.

Streptomyces sp.FJSD 31-2 (CGMCC 4.7321) has genome length of about 11.6Mb, and produces a series of halogenated products, wherein the halogenated products comprise one halogenated enzyme II type PKS product zunyimycin biosynthesis gene cluster with length of 15462 bp. The biosynthetic gene cluster was analyzed in series, among which was the post-modified halogenase gene.

The method comprises the steps of designing a complete coding region amplification primer of a coding gene according to a sequencing result of a Streptomyces 31-2 genome by using biological software such as primer5.0 and the like, adding restriction enzyme Bgl II and HindIII recognition sites into upstream and downstream primers of a Halo coding gene respectively, amplifying a HaloEF/ER halogenase specific primer by using Streptomyces sp.FJS31-2 genome DNA as a template, purifying a PCR product, cloning a TA, carrying out PCR verification by using a general primer M13F/M13R, carrying out sequencing verification on a positive clone, carrying out double digestion on a strain Halo-T by using restriction enzyme Bgl II and HindIII, connecting double digestion fragments with an expression vector pET-32a subjected to corresponding enzyme digestion and dephosphorylation treatment, carrying out room-temperature reaction for 6h, transforming JM109 competent cells, carrying out PCR identification on a primer bacterium liquid and plasmid specific identification, carrying out sequencing verification, and naming a prokaryotic expression plasmid pET-Halo vector subjected to sequencing verification. And finally, transforming BL21(DE3) Rosetta competent cells by the obtained positive clone bacterium plasmid, carrying out PCR (polymerase chain reaction) identification on specific primer bacterium liquid, and determining the positive clone bacterium.

Cloning a target gene or carrying out PCR identification on bacterial liquid:

20 μ L reaction:

the PCR reaction conditions are as follows:

plasmid of positive clone bacterium pET-halo and 100ng plasmid of pET32a are taken to transform escherichia coli BL21(DE3) Rosetta competent cells, after transformant is cultured overnight and T7 promoter bacterial liquid is identified by PCR, the genetic engineering bacterium containing recombinant plasmid is named as pET-halo BL21 and pET32aBL21 respectively. Taking 50 mu L of each of the 2 genetic engineering bacteria, respectively inoculating the bacteria into 5mL of LB liquid culture medium containing 100 ng/mu L ampicillin at 220r/min, respectively inoculating the bacteria into 100mL of LB liquid culture medium containing 100 ng/mu L ampicillin after culturing at 37 ℃ for 3h at 220r/min, respectively, subpackaging 5mL of the culture into 50mL of sterile centrifuge tubes when culturing at 37 ℃ till OD600 is 0.3, adding IPTG at 1.0mmol/L, and placing the tubes on a constant temperature shaking table at 18 ℃, 28 ℃ and 37 ℃ for inducing for 6 h. And (3) centrifuging 1mL of the culture after induction for 5min at 4000r/min, adding 100 mu L of 5 xSDS-PAGE electrophoresis sample buffer solution into the thalli sediment, placing the thalli sediment in a metal bath at 99 ℃ for cracking for 10min at 12000r/min, and centrifuging the thalli sediment for 10min at room temperature to obtain the supernatant, namely the thalli total protein lysate. mu.L of each total protein lysate was loaded and run on a 10% SDS-PAGE gel. After the Coomassie brilliant blue R250 is dyed for 6 hours, the methanol-glacial acetic acid decoloration solution is decolored for 6 hours, and the results show that the expression of the halogenating enzyme protein has no great difference at three different temperatures.

Protein sample processing procedure

(1) Taking 1mL of induced thallus at 12000rpm, centrifuging for 3min, and discarding the supernatant;

(2) adding 1mL of deionized water into each tube, blowing, uniformly mixing, centrifuging at 12000rpm for 3min, and removing the supernatant;

(3) adding 50 μ L deionized water into each tube, blowing, mixing, adding 20 μ L5 xSDS-PAGE electrophoresis sample buffer, and performing metal bath lysis at 99 deg.C for 10 min;

(4) and taking out the centrifuge tube, centrifuging at 12000rpm for 10min to obtain a total protein lysate sample.

SDS-PAGE protein electrophoretic analysis step

(1) Respectively taking 10 mu L of total protein lysate, 10 mu L of protein molecule standard sample application, 10% SDS-PAGE gel electrophoresis analysis, electrophoresis at 50V for 30min, electrophoresis at 100V for 2h, and adjusting time according to actual conditions;

(2) stripping the gel, placing the gel in a protein staining solution containing Coomassie brilliant blue R250, and carrying out shaking staining on a decoloring shaker for 2-6 h;

(3) and decoloring the Coomassie brilliant blue protein decoloring solution for 6h, exposing and developing by a two-color infrared laser imaging system, and judging whether the protein is expressed or not.

TABLE 1 primer sequences and uses

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Sequence listing

<110> Zunyi university of medical science

<120> preparation method and application of halogenase recombinant expression vector

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agctacttcc agggctgcga cttcatcgag ggcgagaacc gggccggcga cgtggtcgtg 600

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ggcacggtca gcatcggcta tgtcacccgc accgctctgc tgcagcgctc cggtctgacc 720

ccggaccagc tgttcgaaca gcagctggcc gcctcggacg aggtcaagcg gctcacccgg 780

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