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Expression method, special expression vector, engineering bacteria and application for recombinant human insulin

A recombinant human insulin and expression vector technology, applied in the biological field, can solve the problems of not being able to have a selection marker-resistance gene, disrupting the balance of the intestinal micro-ecosystem, and adversely affecting normal flora

Active Publication Date: 2015-07-29
WUHAN ZHENFU PHARMA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to fully comply with the true food-grade lactic acid bacteria engineering bacteria, in addition to the food-grade host bacteria, the vectors used must also meet the food-grade requirements, that is, they cannot have the screening markers used by most expression vectors— Resistance genes, because due to the existence of resistance genes, it is possible to have adverse effects on the normal flora of the intestinal tract of animals or humans, disrupting the balance of their normal intestinal micro-ecological system

Method used

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  • Expression method, special expression vector, engineering bacteria and application for recombinant human insulin
  • Expression method, special expression vector, engineering bacteria and application for recombinant human insulin
  • Expression method, special expression vector, engineering bacteria and application for recombinant human insulin

Examples

Experimental program
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Embodiment 1

[0035] Example 1: Synthesis of the recombinant human insulin coding gene. In order to ensure the accuracy of the final coding sequence and the smooth development of subsequent experiments, the recombinant human insulin coding gene (SEQ ID NO: 1) was commissioned to be synthesized by Shanghai Sangon Biotechnology Co., Ltd., and the obtained product contained The plasmid pMD18T-INS of the recombinant gene.

Embodiment 2

[0036] Example 2: Construction of Recombinant Human Insulin Inducible Surface Display Expression Vector and Engineering Bacteria

[0037] see figure 1 Constructing recombinant human insulin inducible surface display expression vector, the specific process includes the following steps:

[0038] 1. Construction of vector pMRF01 containing recombinant human insulin coding sequence

[0039] Amplification primers were designed according to the recombinant human insulin coding sequence, and NcoI and KpnI restriction sites were introduced into the upstream and downstream primers respectively. The primer sequences are as follows:

[0040] Upstream primer P1: 5'-CATG CCATGG CATTTGTTAACCAACATTTATGTGGTTCAC-3' (the underlined part is the NcoI recognition site, and the italic part is the matching base introduced without avoiding the change of the reading frame)

[0041] Downstream primer P2: 5'-GG GGTACC GTTACAGTAGTTCTCAAGTTGATATAATGAACA-3' (the underlined part is the KpnI recognitio...

Embodiment 3

[0055] Example 3: Cell wall display expression of recombinant human insulin in lactic acid bacteria and identification of expression products

[0056] 1. Induced expression of recombinant human insulin in lactic acid bacteria

[0057] Inoculate the corresponding expression engineered bacteria L.lactisNZ3900 / pMRF5018 successfully constructed in Example 2 into 5ml of LM17 medium, culture at 30°C overnight, and inoculate the culture into freshly prepared LM17 at a ratio of 1:50 the next day cultured at 30°C till OD 600 About 0.2-0.5, then add nisin (Nisin) with a final concentration of about 0.1-100ng / ml to the culture for induction, and culture at 30°C for 3-12 hours. At the same time, the L.lactisNZ3900 empty strain and the expression engineering bacteria L.lactisNZ3900 / pMRF5018 were not induced as two groups of control groups.

[0058] The bacterial cell pellet was collected by centrifugation, and was washed with pre-cooled PBS buffer (NaCl 137mmol / L, KCl2.7mmol / L, NaCl 2 H...

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Abstract

The invention provides an expression method, a special expression vector, engineering bacteria and an application for recombinant human insulin. According to the expression method, recombinant human insulin genes can be subjected to induced expression in food-grade lactobacillus, and the recombinant human insulin is presented on the surface of the lactobacillus. The engineering bacteria, the expression vector and an inducer, which are used in the expression method, all meet food-grade requirements, so that the potential hazard caused by existence of resistance genes of the expression vector and non-food-grade inducers is avoided. Lactobacillus engineering bacteria containing cell-wall presentation insulin obtained according to the invention can stimulate an NOD (Non-obese diabetic) rat to produce specific antibodies for the recombinant human insulin, so that the level of a cell factor IL-4 related to immune tolerance is obviously increased, and the generation of immune tolerance is induced; a microbial agent can be taken as an oral vaccine for type I diabetes mellitus as long as the induced lactobacillus engineering bacteria are prepared into the microbial agent, complicated post-processing processes, such as purification, are unnecessary, and wide application prospects are obtained.

Description

【Technical field】 [0001] The invention relates to the field of biotechnology, in particular to a method for expressing recombinant human insulin, a special expression vector, engineering bacteria and its application. 【Background technique】 [0002] Diabetes is a chronic disease caused by insulin deficiency or deficiency caused by congenital or acquired factors. This lack of insulin leads to elevated blood sugar levels which can lead to damage to the body, especially blood vessels and nerves. Polydipsia, polyuria, polyphagia, and weight loss are typical symptoms of diabetes. In severe cases, ketone poisoning and chronic diseases of various organs may also occur. [0003] There are two main types of diabetes: type I and type II. Type I diabetes (Type I diabetes mellitus, T1DM) is an autoimmune disease. The pancreas of the patient is destroyed by the immune system, resulting in an absolute lack of insulin. Blood sugar is stable. Type II diabetes (Type II diabetes mellitus, ...

Claims

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Application Information

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IPC IPC(8): C12N15/17C12N15/74C12N1/21C12P21/02A61K38/28A61P3/10C12R1/46
Inventor 王业富毛瑞峰
Owner WUHAN ZHENFU PHARMA CO LTD
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