Expression method of recombinant human insulin, special expression vector, engineering bacteria and application
A recombinant human insulin and expression vector technology, applied in the biological field, can solve the problems of disturbing the balance of the intestinal micro-ecosystem, not being able to have a selection marker-resistance gene, adverse effects on normal flora, etc.
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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 it overnight at 30°C, and inoculate the culture into freshly prepared LM17 at a ratio of 1:50 the next day , cultured statically at 30°C to 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, KCl2.7mmo...
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