Narcissus tazetta lectin expressed in vitro and application thereof
An in vitro expression and lectin technology, applied in the application and use of vectors to introduce foreign genetic material, plant peptides, etc., can solve the problems of no research reports on the preparation of Chinese narcissus lectin, and achieve the effect of reducing costs
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Embodiment 1
[0017] Example 1: Obtaining the NTL sequence of the Chinese narcissus lectin gene
[0018] The young and fresh leaves of Chinese narcissus (Narcissus tazetta var. Chinensis) were used as materials to extract mRNA and reverse transcribe it into cDNA as a template. Primers were designed according to the published conserved sequences of the plant lectin gene family, and the coding sequence was amplified by PCR. PCR reaction system (20 μL): 2×GC Buffer I 10 μL, dNTP (each 2.5 mmol·L -1 ) 1.6 μL, upstream primer (10 μmol L -1 ) 2 μL, downstream primer (10 μmol L -1 )2μL, cDNA (≈0.04μg·L -1 ) 1 μL, LA Taq enzyme (5U·μL -1 ) 0.2 μL, ultrapure water 3.2 μL.
[0019] Upstream primer (as shown in SEQ ID No.6):
[0020] 5′-TCATTCTGGCCACCATCTTC-3′
[0021] Downstream primer (as shown in SEQ ID No.7):
[0022] 5′-TGGCGGTCACTAACTTTATC-3′
[0023] Perform agarose gel electrophoresis detection on the PCR amplification product, cut out the target band, purify and recover, connect the r...
Embodiment 2
[0038] Embodiment 2 Construction of the recombinant expression vector carrying NTL gene
[0039] Using the Chinese Narcissus cDNA as a template, design primers according to SEQ ID No.1, and PCR amplify the deoxyribonucleotide sequence of the mature protein encoded by the Chinese Narcissus lectin gene. PCR reaction system (20 μL): 10×GC Buffer 2 μL, dNTP (each 2.5 mmol·L -1 ) 1.6 μL, upstream primer (10 μmol L -1 ) 2 μL, downstream primer (10 μmol L -1 )2μL, cDNA (≈0.04μg·L -1 ) 1 μL, Probest DNA polymerase (5U·μL -1 ) 0.1 μL, ultrapure water 11.3 μL. The two ends of the primers were respectively introduced with protective bases and enzyme cutting sites (BamH I and Not I). The primer sequences are as follows:
[0040] Upstream: 5′-TT GGATCC ATGGACAACAACATTCTCTACTC-3', the underlined part is the restriction site of BamH I, as shown in SEQ ID No.4.
[0041] Downstream: 5′-AAT GCGGCCGC TTACTTGGCGGTCACTAA-3', the underlined part is the Not I restriction site, as shown in ...
Embodiment 3
[0044] Example 3 Recombinant Expression Vector Transformation Host, Induced Expression and Purification
[0045] 1) Recombinant expression vector transforms the host and induces expression
[0046] The recombinant expression vector pGEX-NTL constructed in Example 2 was transformed into Escherichia coli BL21(DE3) competent cells, and single colonies grown on the Amp resistant plate were picked for PCR identification, and positive clones were used to induce expression. Correctly identified clones were cultured overnight, and then the bacteria were diluted 100-fold in ampicillin-containing (100 mg·L -1 ) in the LB medium, until the bacterial solution grows to OD 600 When it is 0.6-0.8, add IPTG (final concentration 1mmol L -1 ) for induction culture.
[0047] 2) Protein extraction and purification
[0048] ①Collect the induced bacterial liquid and centrifuge at 10,000g for 10min to collect the bacterial cells; ②Add the binding solution to suspend the cells (300mM NaCl, 50mM s...
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