CN111234028B - Small molecule protein for indicating calcium ions and application thereof - Google Patents

Small molecule protein for indicating calcium ions and application thereof Download PDF

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CN111234028B
CN111234028B CN201811435510.6A CN201811435510A CN111234028B CN 111234028 B CN111234028 B CN 111234028B CN 201811435510 A CN201811435510 A CN 201811435510A CN 111234028 B CN111234028 B CN 111234028B
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calcium
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molecule protein
protein
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CN111234028A (en
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李翔
杨海洋
钱政江
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Shenzhen Institute of Advanced Technology of CAS
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Abstract

The invention provides a small molecular protein for indicating calcium ions and application thereof, wherein the amino acid sequence of the small molecular protein is shown as SEQ ID NO. 1; the gene coded small molecular protein provided by the invention is named as GEM, is fused with calmodulin (CaM) and myosin light chain kinase M13, can overcome a plurality of defects in the existing calcium imaging technology, can realize a novel calcium imaging technology which gives consideration to the whole situation and the microcosmic situation and can record the activity in the experimental animal brain which can move freely in the true sense by adopting the small molecular protein provided by the invention, and has wide application prospect and great market value.

Description

Small molecule protein for indicating calcium ions and application thereof
Technical Field
The invention relates to the field of biology, in particular to a small molecular protein for indicating calcium ions and application thereof.
Background
Calcium imaging technology (calcium imaging) refers to a method of measuring the concentration of calcium ions in tissue cells using calcium ion indicators. In living organisms, calcium ions generate various intracellular signals, which are present in almost every type of cell and play important roles in many functions, such as control of myocardial cell contraction, and the like. Calcium ions are an important class of neuronal intracellular signaling molecules in the mammalian nervous system. In resting state, most neurons have intracellular calcium ion concentration of about 50-100nM, which can rise 10-100 times when neurons are excited, and is essential for neurotransmitter release. In general, the activity of neurons is closely related to the calcium ion concentration in the neurons, and the neurons explode a short calcium ion concentration peak when discharging. According to the strict corresponding relation between the calcium ion concentration and the neuron activity, the calcium ion concentration in the neuron is expressed by the fluorescence intensity by using a special fluorescent dye or a protein fluorescent probe, so that the purpose of detecting the neuron activity can be achieved, and the technology is a neuron calcium imaging technology.
Currently, there are two main categories of calcium ion indicators used for calcium imaging: one is a chemical calcium ion indicator. Another class is gene-encoded calcium ion indicators. Chemical calcium ion indicators generally refer to small molecules that can chelate calcium ions. The calcium ion indicator encoded by the current gene is mainly a fluorescent protein from Green Fluorescent Protein (GFP) and variants thereof (such as YFP, CFP and the like), and is fused with calmodulin (CaM) and myosin light chain kinase M13. At present, the calcium ion indicators coded by more widely used genes comprise: GCaMP, pericams, TN-XXL and the like, wherein GCaMP is widely applied to in-vivo calcium imaging research at present due to the super-strong sensitivity of GCaMP.
CN101372482 discloses a fluorescein calcium ion fluorescent indicator, wherein R1 and R2 are selected from hydrogen, methyl, ethyl, propyl or isopropyl, a synthesized fluorescein calcium ion indicator crude product is esterified with anhydrous methanol to obtain a fluorescein calcium ion indicator methyl ester, an esterified pure product is obtained by column chromatography separation, and finally, the esterified pure product is hydrolyzed to obtain the fluorescein calcium ion fluorescent indicator pure product. The fluorescein calcium ion fluorescent indicator can be used for measuring the concentration of intracellular free calcium ions. Currently, the recording process of imaging is very similar regardless of which type of calcium ion indicator is used. I.e., cells with calcium ion indicator can be observed by fluorescence microscopy and then images captured and recorded by a CCD camera. Due to the limitations of ex vivo experiments, more and more researchers such as neuroscientists are inclined to obtain in vivo calcium imaging experiments because in vivo experiments can more accurately reflect data of physiological conditions. In order to achieve the purpose of calcium imaging in vivo in real time, the calcium ion imaging technology must be completed by a two-photon microscope. Although two-photon microscopy has progressed rapidly in these years, it has several very fatal drawbacks:
firstly, the operation is very complicated, when calcium imaging is combined with two-photon observation of calcium activity in the brain of an experimental animal, the experimental animal needs to be processed in advance, for example, skull of the animal is polished, so as to achieve a better imaging effect; second, existing calcium imaging techniques are limited to the field of view of the microscope and only record a small area of the brain. The normal operation of the nervous system is realized by the mutual cooperation of a plurality of different brain areas, so the research on the processes needs to carry out detailed and comprehensive observation and analysis on the whole central nervous system and even the peripheral nervous system, and the current calcium imaging technology does not meet the requirement; thirdly, because the imaging of the existing calcium imaging technology needs a two-photon microscope, the optical path of the imaging is very stable and single, scientific research personnel are required to fix the experimental animal for imaging, and in this case, the activity in the brain is greatly different from the activity in the brain of the animal in a normal free state, and the obtained data is also inaccurate.
Therefore, the field of neuroscience urgently needs a calcium imaging technology which can take account of the whole situation and the microcosmic situation and can record the activity in the brain of the experimental animal which can move freely in the real sense, develops a novel gene-coded small molecular protein which is used as a calcium ion indicator and has wide application prospect and huge market value.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides a small molecule protein for indicating calcium ions and application thereof, wherein the small molecule protein can overcome a plurality of defects in the prior calcium imaging technology by fusing with calmodulin (CaM) and myosin light chain kinase M13 (namely, GEM is used for replacing fluorescent protein in the prior GCaMP), and has wide application prospect and great market value as a calcium ion indicator.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a small molecule protein for indicating calcium ions, and the amino acid sequence of the small molecule protein is shown as SEQ ID NO. 1.
The SEQ ID NO.1 is as follows:
MRRRQRASRAGVVARRATPGDAQANSGRAPARPFQSFVSNGAMLMNKQIVAGIVAGLVSMSSHAQLGQLFQSVKEQVTQAATSQVNQGVRSATDEAVQATSSRTRKAINSVRSPSSAAAATSTSPSAAEETNDATLSEARK.
preferably, the affinity metal ion of the small molecule protein comprises a manganese ion of valency 2.
Preferably, the nucleotide sequence of the small molecule protein is shown as SEQ ID NO. 2:
ATGGGTGTAGTCGCACGAAGAGCTACACCCGGTGACGCACAAGCCAACTCAGGTAGAGCTCCCGCCCGGCCATTTCAGTCTTTTGTGTCAAACGGAGCCATGCTGATGAACAAGCAAATTGTGGCTGGGATAGTTGCAGGTCTGGTCAGCATGTCCTCCCACGCACAGCTCGGTCAACTCTTCCAGTCTGTTAAGGAACAGGTCACTCAGGCAGCAACCTCTCAGGTAAATCAAGGAGTAAGGTCCGCTACTGACGAAGCCGTGCAAGCAACATCTAGTCGGACCAGAAAGGCCATTAACAGTGTTAGGTCCCCCTCAAGCGCAGCAGCCGCTACTTCTACAAGCCCTTCTGCTGCCGAGGAGACTAATGACGCTACCCTGAGTGAGGCCCGCAAATAA.
preferably, the small molecule protein has a molecular weight of less than 15KD.
According to the invention, the small molecular protein can be combined with manganese ions in vivo through complex experimental verification, the manganese ions are paramagnetic substances, and the GEM protein combined with the manganese ions presents high signals under a T1 weighted image during Magnetic Resonance Imaging (MRI).
The invention claims the gene sequence, but not limited to, and the gene mutation and modification made on the basis of the gene sequence belong to the protection points of the invention.
In a second aspect, the present invention provides a plasmid comprising the nucleotide sequence of the small molecule protein of the first aspect.
In a third aspect, the invention provides a lentivirus obtainable by co-transfection and packaging of the plasmid of the second aspect and a helper plasmid.
In a fourth aspect, the invention provides a use of the small molecule protein of the first aspect, the plasmid of the second aspect, or the lentivirus of the third aspect for indicating calcium ions.
The invention provides a gene coded indicator, which is a DNA tool in nature although functioning as a protein, and the expression of the protein is automatically completed in an organism.
In a fifth aspect, the present invention provides a calcium ion imaging method, wherein the method employs any one or a combination of at least two of the small molecule protein of the first aspect, the plasmid of the second aspect, or the lentivirus of the third aspect for magnetic resonance imaging.
The invention provides a gene-coded small-molecule protein named as GEM, which is fused with calmodulin (CaM) and myosin light chain kinase M13 (namely GEM is used for replacing fluorescent protein in the prior GCaMP), can overcome a plurality of defects in the prior calcium imaging technology, and realizes a calcium imaging technology which can give consideration to both global and microscopic aspects and can record the activity in the brain of an experimental animal which can really freely move.
The invention is not limited to a certain form of fusion transformation, and all the calcium ion imaging technology developed on the basis of the fusion transformation belongs to the protection scope of the invention
The application of the present invention is not limited to the application of the central nervous system, and the application of the peripheral nervous system, the blood circulation system, etc. are all within the scope of the present invention.
Compared with the prior art, the invention has the following beneficial effects:
the small molecular protein provided by the invention has definite gene sequence and small molecular weight, and is easier to operate for subsequent optimization and improvement; the invention utilizes the nuclear magnetic resonance technology to image the change of calcium ions, overcomes the limitation of the field of view of a two-photon microscope in the prior calcium imaging technology, and can detect the electrical activity of neurons in the brain globally and microscopically; the invention does not need to fix the experimental animal when carrying out calcium imaging, can really realize the electrical activity of the neuron in the Free-moving state, and has more accurate data; because the nuclear magnetic imaging technology has no limitation of tissue penetrability, the experimental animal does not need to be pretreated, and the experimental period, manpower and material resources and the like are greatly saved.
Drawings
FIG. 1 is a schematic view of the structure of M13-GEM-CaM of the present invention;
fig. 2 is a 7.0T MRI scan result of the present invention, in which fig. 2 (a) is a T1 imaging result graph of a coronal plane, fig. 2 (B) is a T2 imaging result graph of a coronal plane, fig. 2 (C) is a T1 imaging result graph of a horizontal plane, fig. 2 (D) is a T2 imaging result graph of a horizontal plane, fig. 2 (E) is a T1 imaging result graph of a sagittal plane, and fig. 2 (F) is a T2 imaging result graph of a sagittal plane.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.
Example 1 plasmid construction
A GEM core nucleotide sequence;
ATGGGTGTAGTCGCACGAAGAGCTACACCCGGTGACGCACAAGCCAACTCAGGTAGAGCTCCCGCCCGGCCATTTCAGTCTTTTGTGTCAAACGGAGCCATGCTGATGAACAAGCAAATTGTGGCTGGGATAGTTGCAGGTCTGGTCAGCATGTCCTCCCACGCACAGCTCGGTCAACTCTTCCAGTCTGTTAAGGAACAGGTCACTCAGGCAGCAACCTCTCAGGTAAATCAAGGAGTAAGGTCCGCTACTGACGAAGCCGTGCAAGCAACATCTAGTCGGACCAGAAAGGCCATTAACAGTGTTAGGTCCCCCTCAAGCGCAGCAGCCGCTACTTCTACAAGCCCTTCTGCTGCCGAGGAGACTAATGACGCTACCCTGAGTGAGGCCCGCAAATAA
the DNA sequence is a core sequence after design and modification, and can combine manganese ions in an animal body after verification, and a high signal can be presented under nuclear magnetic resonance imaging; because the DNA sequence is designed and modified, the DNA sequence needs to be obtained by artificial synthesis;
universal calmodulin CaM and myosin light chain M13DNA sequences were synthesized and ligated to the C-and N-termini of the core sequence GEM, see fig. 1; this pattern is the classical structural pattern of GCaMP, which has been verified to encode calcium ion indicators for the most commonly used genes at present;
the invention utilizes the original structural framework and replaces the original fluorescent sequence GFP with the GEM sequence which is designed by modification, so the signal effect of the MRI of the GEM can be verified in the invention; in FIG. 1, calmodulin CaM is a protein capable of reacting with calciumThe ion-binding protein, myosin light chain M13, is capable of binding to calmodulin GaM, when calcium ions are bound in vivo, due to Ca 2+ -CaM-M13 interaction, the conformational change leading to a conformational change in the core protein GEM, which in turn leads to a change in its signal upon MRI imaging, allowing monitoring of changes in the calcium ion level in vivo.
Construction of the complete plasmid: inserting M13-GEM-CaM into a vector pET32a, wherein the enzyme cutting sites are respectively selected to be NcoI/NotI, wherein the core function of the invention is not influenced by the selection of the vector and the selection of the enzyme cutting sites, and the selection can be carried out according to different actual requirements.
Example 2 Experimental testing
Intracerebral virus microinjection:
the rat striatum brain area is selected for verification (the nucleus mass of the striatum is relatively large, uniform and good in isotropy, and other brain areas can be used as experimental brain areas); after adult rats are anesthetized by chloral hydrate intraperitoneal injection, the rats are fixed on a three-dimensional position in a prone mode, and the periosteum is cut and separated in the middle of the scalp; according to the position (A/P0.6, M/L3.0, D/V5.2) of a striatum nucleus in a rat brain atlas, a small hole is formed in the skull by a cranial drill, 2000nL of lentivirus is slowly injected into a striatum brain area by a 10 microliter micro injection needle at the sampling speed of 100nL/min, after the injection is finished, the needle is stopped for 5min, and the needle head is slowly removed;
after 5-6 weeks after virus injection, rats were anesthetized and then scanned for MRI (uMR, shanghai United Imaging Healthcare), using a three-dimensional magnetization pre-gradient echo sequence with a voxel resolution of 0.25 × 1.5mm, and T1-weighted images of the coronal and horizontal planes of the rat brain were collected for 4-6 minutes each.
The results are shown in fig. 2 (a) -2 (F), GEM can and only presents signals under T1-weighted imaging.
In conclusion, the invention provides a small molecular protein for indicating calcium ions and application thereof, named as GEM, which is fused with calmodulin (CaM) and myosin light chain kinase M13, can overcome a plurality of defects in the existing calcium imaging technology.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Figure RE-IDA0001934810210000011
Figure RE-IDA0001934810210000021
Figure RE-IDA0001934810210000031

Claims (5)

1. The small molecule protein for indicating calcium ions is characterized in that the amino acid sequence of the small molecule protein is a sequence coded by a nucleic acid sequence shown in SEQ ID NO. 2.
2. A plasmid comprising a nucleotide sequence encoding the small molecule protein of claim 1.
3. A lentivirus packaged by cotransfection of the plasmid of claim 2 and a helper plasmid.
4. An indicator calcium ion for use in non-disease diagnostic and therapeutic purposes comprising the small molecule protein of claim 1, the plasmid of claim 2, or the lentivirus of claim 3.
5. A method of calcium ion imaging for non-disease diagnosis and therapy, wherein the method employs any one or a combination of at least two of the small molecule protein of claim 1, the plasmid of claim 2, or the lentivirus of claim 3 for magnetic resonance imaging.
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