阅读说明：本技术 一种对绿光敏感的光敏蛋白质粒的设计方法 (Design method of photosensitive protein plasmid sensitive to green light ) 是由 郑斌 郭明明 明东 甘霖 刘爽 于 2020-05-08 设计创作，主要内容包括：本发明公开一种对绿光敏感的光敏蛋白质粒的设计方法,主要步骤包括:制备上转换纳米颗粒；寻找合适的NpHR基因序列,合成NpHR,构建质粒；检测重组质粒的表达情况,检测NpHR；用AAV包装将连接好的质粒,检测包装情况；用膜片钳检测细胞膜两侧的电位变化。以往的神经疾病的治疗效果差,并需要电极的植入,危险性高。本发明设计了一种可在脑内表达的光敏蛋白,与上转换纳米颗粒同注射进入脑内,在近红外照射下,上转换纳米颗粒可发出绿光,绿光可激发光敏蛋白通道打开,使阴离子内流,从而抑制神经元的兴奋。(The invention discloses a design method of photosensitive protein plasmid sensitive to green light, which mainly comprises the steps of preparing up-conversion nano particles; searching a proper NpHR gene sequence, synthesizing NpHR, and constructing a plasmid; detecting the expression condition of the recombinant plasmid, and detecting NpHR; packaging the connected plasmids by AAV, and detecting the packaging condition; the potential change on both sides of the cell membrane was detected with patch clamp. The conventional treatment of neurological diseases is poor in effect, requires implantation of an electrode, and is highly dangerous. The invention designs photosensitive protein capable of being expressed in brain, the photosensitive protein and the up-conversion nano particles are injected into the brain together, under the irradiation of near infrared rays, the up-conversion nano particles can emit green light, the green light can excite the photosensitive protein channel to be opened, so that anions can flow in, and the excitation of neurons is inhibited.)

1. A design method of a photosensitive protein plasmid sensitive to green light is characterized by comprising the following steps:

1) preparing up-conversion nanoparticles;

2) searching a proper NpHR gene sequence, synthesizing NpHR, and constructing a plasmid hSyn-eNPHR-eYFP;

3) detecting the expression condition of the recombinant plasmid, and detecting NpHR;

4) packaging the connected plasmids by AAV, and detecting the packaging condition;

5) the potential change on both sides of the cell membrane was detected with patch clamp.

2. The method for designing photosensitive protein plasmid sensitive to green light according to claim 1, wherein the step 3) constructs plasmid hSyn-eNpHR-eYFP.

3. The method for designing a photosensitive protein plasmid sensitive to green light according to claim 1, wherein the step 3) is performed to construct the plasmid mCaMK IIa-eNPHR-eYFP.

4. The method for designing a photosensitive protein plasmid sensitive to green light according to claim 1, wherein the step 3) is to construct the plasmid hSyn-eNpHR-mCherry.

5. The method of claim 1, wherein the NpHR gene sequence:

Atcatcagccggggtcccagaagcagatggaacatccaaaatgctgccggacacgacagattcgtttgacgtcagatagttcaggagcaagaatgcgaaaatgtactttgccacgatatccaggaaagaatatccccagctcgtcaccccaacgggcaacaccgcgataccctcgactccaagagcccacacaattggataccccagccacatcactactgtcaacagcttcagggtattgaacatgtcagcggttcccgcggctttggcgtcctgtgcccactccaccagcaggatatacaagaccaccagaaagcatgcacaactgatagcgtaccagaaccatctcatgaggtgggaggaggtagtcagggccgcggcaaggccagtcacgcacatagcgatatcgaaagtgatagctgtgaagagctttgtagcattgcttccggccaggagacccagagcgaggagaatcatgggcgtggaaagtgcccaggtgagataccgtccccacatggtgactacaccatctacctcttctcctcccagcatcactgagctgccttctgcaaaatggcccgctggcatttcaagaacggaaattgtcaggccgctcgccaatccagtgtaggaggcaatgctgacgacaggcacaaggatggttgacacagcaataagttttgcccgtggatcatcgagtcctcgggtcataaaaacgaacagcagtatactcagtcctgcaagtgcgatgttgatatagagactgcttgcaagcaaagggtcgttcagcacgaactcgaacaactccctttgggtaacctcggcttgaagggccacggcactct。

Technical Field

The invention belongs to the field of biomedical materials, and relates to a design method for designing photosensitive protein plasmids sensitive to green light, which can be specifically expressed on cell membranes under the irradiation of the green light and can open ion channels on the cell membranes.

Background

Parkinson's Disease (PD) is a common nervous system degenerative disease, and is common in the elderly, with the average age of about 60 years, and the onset of juvenile Parkinson's disease below 40 years being rare. The prevalence rate of PD in people over 65 years old in China is about 1.7%. Most parkinson's disease patients are sporadic cases, with less than 10% of patients having a family history. The most prominent pathological change of parkinson's disease is the degenerative death of mesocerebral Dopaminergic (DA) neurons, which causes a marked reduction in striatal DA content and causes disease.

Optogenetics generally refers to techniques that combine optical and genetic means to precisely control specific neuronal activity. Introducing exogenous photosensitive protein gene into living cell by means of molecular biology, virus biology and the like, and expressing photosensitive channel protein on a cell membrane structure; then controlling the activation and the closing of the photosensitive channel protein on the cell membrane structure by the irradiation of light with specific wavelength; activation and closure of the photoperions can control the opening and closing of ion channels on cell membranes, thereby altering the change in cell membrane voltage, such as depolarization and hyperpolarization of the membrane. When the membrane voltage depolarizes and exceeds a certain threshold value, the neuron is induced to generate a conductive electric signal, namely the neuron is activated; in contrast, when the membrane voltage is hyperpolarized to a certain level, the generation of action potentials of neurons, i.e. the inhibition of neurons, is inhibited. Neuron biologists often use the technology to control the activity of specific neurons by an optical method without damage or with low damage so as to research the function of the neural network, and the technology is particularly suitable for behavioral experiments of in-vivo and even conscious animals.

Current optogenetic methods require the implantation of electrodes into the brain sack, which is highly dangerous and technically demanding, and therefore, the use of upconverting materials can avoid this problem. The upconverting material emits green light under near infrared irradiation, and the designed plasmid is expressed on the cell membrane in the presence of the green light and opens the ion channel.

The method has the following advantages: 1) avoiding the dangerous way of implanting electrodes into the brain; 2) the opening condition of an ion channel on a cell membrane can be artificially controlled; 3) provides a new idea for Parkinson's disease.

Disclosure of Invention

The invention utilizes the upconversion nanometer material and the photosensitive protein NpHR to realize the controllable flow of cell membrane ions, and the NpHR can enable chloride ions to flow in with the help of green light emitted by the upconversion nanometer material, thereby reducing the excitation of neurons and controllably enabling the ions to flow in.

The technical scheme of the invention is a design method of photosensitive protein plasmid sensitive to green light, which comprises the following steps:

1) preparing up-conversion nanoparticles;

2) searching a proper NpHR gene sequence, synthesizing NpHR, and constructing a plasmid hSyn-eNPHR-eYFP;

3) detecting the expression condition of the recombinant plasmid, and detecting NpHR;

4) packaging the connected plasmids by AAV, and detecting the packaging condition;

5) the potential change on both sides of the cell membrane was detected with patch clamp.

And 3) constructing a plasmid hSyn-eNPHR-eYFP.

The step 3) constructs plasmid mCaMK IIa-eNPHR-eYFP.

The step 3) constructs plasmid hSyn-eNPHR-mCherry.

The NpHR gene sequence of the invention is as follows:

Atcatcagccggggtcccagaagcagatggaacatccaaaatgctgccggacacgacagattcgtttgacgtcaga tagttcaggagcaagaatgcgaaaatgtactttgccacgatatccaggaaagaatatccccagctcgtcaccccaacggg caacaccgcgataccctcgactccaagagcccacacaattggataccccagccacatcactactgtcaacagcttcaggg tattgaacatgtcagcggttcccgcggctttggcgtcctgtgcccactccaccagcaggatatacaagaccaccagaaag catgcacaactgatagcgtaccagaaccatctcatgaggtgggaggaggtagtcagggccgcggcaaggccagtcacgca catagcgatatcgaaagtgatagctgtgaagagctttgtagcattgcttccggccaggagacccagagcgaggagaatca tgggcgtggaaagtgcccaggtgagataccgtccccacatggtgactacaccatctacctcttctcctcccagcatcact gagctgccttctgcaaaatggcccgctggcatttcaagaacggaaattgtcaggccgctcgccaatccagtgtaggaggc aatgctgacgacaggcacaaggatggttgacacagcaataagttttgcccgtggatcatcgagtcctcgggtcataaaaa cgaacagcagtatactcagtcctgcaagtgcgatgttgatatagagactgcttgcaagcaaagggtcgttcagcacgaac tcgaacaactccctttgggtaacctcggcttgaagggccacggcactct。

the invention has the advantages that: 1) avoiding the dangerous way of implanting electrodes into the brain; 2) the opening condition of an ion channel on a cell membrane can be artificially controlled; 3) provides a new idea for Parkinson's disease.

The present invention will be further described with reference to the following examples.

Example 1:

preparation of up-conversion nanoparticles and design of plasmids and AAV packaging plasmids. The method comprises the following steps:

1) preparing the upconversion nanoparticles.

2) Searching a proper NpHR gene sequence, synthesizing NpHR, and constructing a plasmid hSyn-eNPHR-eYFP.

3) Detecting the expression of the recombinant plasmid, and detecting the NpHR.

4) The ligated plasmids were packaged with AAV and the packaging was examined.

5) The potential change on both sides of the cell membrane was detected with patch clamp.