Process for preparing anti-hypertension polypeptides by enzymolysis of MRJP1 expression products of China honey bee

A technology of Chinese honeybee and expression products, applied in the fields of botanical equipment and methods, biochemical equipment and methods, animal/human peptides, etc.

Inactive Publication Date: 2008-10-22
ZHEJIANG UNIV
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But so far there has been no relevant report on the use of genetic engineering m

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  • Process for preparing anti-hypertension polypeptides by enzymolysis of MRJP1 expression products of China honey bee
  • Process for preparing anti-hypertension polypeptides by enzymolysis of MRJP1 expression products of China honey bee
  • Process for preparing anti-hypertension polypeptides by enzymolysis of MRJP1 expression products of China honey bee

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Embodiment 1

[0034] The preparation method of the eukaryotic expression product of Apis cerana MRJP1 that promotes cell growth is characterized in that the steps are as follows:

[0035] 1) Cloning of MRJP1 gene

[0036] a) Using the MRJP1 cDNA from the head of 8-day-old worker bees of Apis cerana as a template, using Primer Primer 5.0 software, the sequence of the upstream primer F-Prime was designed as 5'- CCATGG ACATGACAAGGTGGTTGTT-3', the downstream primer R-Prime sequence is 3'TAAAGTTATGTAGACATT CGCCGGCG -5', respectively introduce restriction sites Nco I and Not I into the upstream primer F-Prime sequence and the downstream primer R-Prime sequence (see the underlined part);

[0037] b) The PCR reaction system is 50 μl in total, and the composition is as follows: 5 μl 10×PCR Buffer, 6 μl 25mM MgCl 2 , 1μl 10mM dNTP Mix, 2μl 10μM F-Prime, 2μl 10μM R-Prime, 5μl Apis cerana MRJP1cDNA template, 0.75μl 5U / μl Taq DNA Polymerase, add ddH 2 O to a final volume of 50 μl;

[0038] The PCR ...

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Abstract

The invention discloses a method for preparing antihypertensive polypeptides through zymohydrolysis of an Apis cerana MRJP1 expression product. The method is as follows: firstly, construction of a Bac-to-Bac/BmNPV rhabdovirus expression system is utilized for obtaining recombinant Bacmid-MRJP1 which is then transfected to silkworm cells; secondly, after recombinant viruses are obtained, silkworm larvas are injected; thirdly, after the silkworm larvas are transfected for 96 hours, haemolymph is collected; fourthly, after purification of an affinity column, the high-purity recombinant MRJP1 is obtained; fifthly, the purified Apis cerana MRJP1 expression product is restricted by trypsinase and centrifugally filtered by an ultrafiltration membrane and phosphate is removed by CaCl2, and then antihypertensive polypeptide compounds are obtained. The polypeptides prepared by the method have the function of reducing the blood pressure.

Description

technical field [0001] The invention relates to a method for preparing an antihypertensive polypeptide by enzymatic hydrolysis of an expression product of Apis cerana MRJP1. Background technique [0002] The angiotensin-converting enzyme (ACE) inhibitory peptide was first discovered by Ferreira (1965) from the venom of the American cobra (Bothrops jararaca), and Oshima et al. found the ACE inhibitory peptide from the bacterial collagenase digestion solution of gelatin. Cushman et al. developed the first-generation ACEI antihypertensive drug Captopril based on the ACE binding site hypothesis model, and achieved great success in the clinical treatment of hypertension. Subsequently, the second and third generation chemically synthesized ACE inhibitors were developed, such as Lisinopril, Enalapril, Forsinopril, etc. Most of these antihypertensive drugs are analogs of short-chain peptides. Synthetic ACE inhibitory peptide has the characteristics of fast and strong drug effect, b...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/866C07K14/435C12P21/06A61P9/12
Inventor 苏松坤陶挺缪云根陈盛禄
Owner ZHEJIANG UNIV
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