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The serotonin receptor gene pr5-ht8 of Pieris rapae and its application

A serotonin receptor, pr5-ht8 technology, applied in the fields of application, genetic engineering, receptor/cell surface antigen/cell surface determinant, etc., can solve the problem of less insect research

Inactive Publication Date: 2016-04-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on insect 5-HT receptors mainly focuses on the model insects Drosophila melanogaster and honeybee Apismellifera, and there are relatively few studies on other insects.

Method used

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  • The serotonin receptor gene pr5-ht8 of Pieris rapae and its application
  • The serotonin receptor gene pr5-ht8 of Pieris rapae and its application
  • The serotonin receptor gene pr5-ht8 of Pieris rapae and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Embodiment 1, cabbage caterpillar 5-hydroxytryptamine receptor Pr5-HT 8 clone

[0062] 1. Use the Trizol method to homogenize the larvae of Pieris rapae about 5 instars to extract total RNA and transcribe it into cDNA:

[0063] Reverse transcription experiments were performed using TransScriptTMIIReverseTranscriptase.

[0064] reaction system:

[0065] Total RNA (1μg / μl) 1ul

[0066] dNTP Mixture (10mMeach) 1ul

[0067] Random6mers (20uM) 1ul

[0068] RNaseFreedH 2 O7ul;

[0069] Remarks: Random6mers are random 6 nucleotide primers;

[0070] Reaction conditions: 65°C, 5 minutes, immediately place on ice for 2 minutes, then add the following components:

[0071] 5×PrimeScriptRTBuffer (5 times PrimeScript reverse transcription buffer) 4ul

[0072] RNase Inhibitor (40U / ul) (RNase inhibitor) 0.5ul

[0073] TransScriptTMIIReverseTranscriptase (TransScriptTMII Reverse Transcriptase) 0.5ul

[0074] RNaseFreedH 2 O5ul

[0075] Reaction conditions: 30°C, 10min, 42°C...

Embodiment 2

[0092] Embodiment 2, cabbage caterpillar 5-hydroxytryptamine receptor protein Pr5-HT 8 Expressed in HEK-293 cell line

[0093] 1. The cloned Pieris rapae Pr5-HT 8 The gene sequence (SEQ ID NO: 1) was cloned into the pcDNA3 expression vector:

[0094] Preparation of pcDNA3 expression vector: EcoR1 and Kpn1 double digestion reaction system

[0095] pCDNA3 (200ng / ul) 41ul

[0096] 10×MBuffer5ul

[0097] EcoR1 (10U / ul) 2ul

[0098] Kpn1(10U / ul)2ul

[0099] Reaction conditions: 37°C 4hours;

[0100] Use TaKaRaAgaroseGelDNAPurificationKit to cut the gel and recover the above-mentioned digested products.

[0101] Use ligase (TaKaRaT4DNALigase) to connect the sequence obtained in Example 1 to the above enzyme-cut vector to obtain the expression vector pcDNA3-Pr5-HT 8 .

[0102] 2. Transfection of HEK-293 cell line and monoclonal cell culture

[0103] pcDNA3-Pr5-HT 8 HEK-293 cell line was transfected with lipofectamine2000 liposome transfection reagent, and the transfection ...

Embodiment 3

[0106] Example 3, HEK-Pr5-HT 8 Calcium ion responses of cell lines to various biogenic amines and 5-HT receptor agonists and antagonists

[0107] will express Pr5-HT 8 (i.e. the pcDNA3-Pr5-HT obtained in Example 2 8 ) Cells were transferred to 12mm coverslips 48 hours in advance, washed twice with DPBS (Dulbeccophosphate-buffered saline, Dulbecco's phosphate-buffered saline) and then treated with Fura-2AM (calcium fluorescent probe, final concentration 5M) and DMEM (Dulbecco's modification of Eagle's medium Dulbecco , modified Duchenne’s Eagle’s medium) for 30 min, then washed twice with Ringerbuffer, and then measured the fluorescence intensity at 510 nm emission wavelength at 340 nm and 380 nm excitation wavelength respectively on the ERP calcium ion imaging system of PTI company. The ratio can be converted into the absolute concentration of intracellular calcium ions.

[0108] Remarks: HEK-293 and HEK-Pr5-HT 8 There is basically no difference in the calcium ion concentr...

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Abstract

The invention relates to the fields of the molecular biology, the cell biology and the agricultural pharmacology, mainly relates to a novel 5-hydroxytryptamine acceptor gene expressed in cabbage caterpillar, and its coding protein, and also relates to a use of the protein and the acceptor gene in the detection of the screening and / or the evaluation of substances for regulating the activity of a 5-hydroxytryptamine acceptor. The above detection method sequentially comprises the following steps: 1, transferring a recombinant vector containing the cabbage caterpillar 5-hydroxytryptamine acceptor gene Pr5-HT8 to a host cell line; 2, processing cells obtained in step 1 by a candidate substance; and 3, detecting the change of the concentration of calcium ions in cells obtained in step 2, wherein the rise of the concentration of the calcium ions in the cells shows that the candidate substance can activate the expressed target acceptor Pr5-HT8.

Description

technical field [0001] The invention relates to the fields of molecular biology, cell biology and pesticide science, and mainly relates to a novel 5-hydroxytryptamine receptor gene expressed in cabbage caterpillar and its coded protein; the invention also relates to the application of the protein and the receptor gene. Background technique [0002] 5-hydroxytryptamine (5-hydroxytryptamine, 5-HT) is an important biogenic amine in many animals, it can be used as a neurotransmitter, neuromodulator or neurohormone to control and regulate many important physiological and behavioral processes, such as eel lampreys Movement behavior (Harris-Warrick and Cohen1985), aggressive behavior of the lobster Homarusamericanus (Kravitz2000), insect reproduction (Lange2004), aggregation (Ansteye et al.2009), learning and memory, etc. (Sitaraman et al.2008). 5-HT exerts different physiological functions, many of which are realized through its receptors. One potential significance of the study ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/705C12N15/85C12Q1/02
Inventor 叶恭银齐易香黄佳
Owner ZHEJIANG UNIV
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