Protein gene of hepatitis C virus F and use thereof
A hepatitis C virus and protein technology, applied in the fields of genetic engineering and biomedical diagnostics, can solve the problems of inability to guarantee blood safety, poor sensitivity and specificity, missed detection and false detection.
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Embodiment 1
[0077] Example 1F protein gene codon replacement
[0078] Referring to the HCV type 1b C gene sequence (GI: 329737), the 10th codon of the C gene was determined to be the 1st codon of the F gene, and after the first codon was read according to the C+1 reading frame until the stop codon, we obtained Wild-type F gene sequence (as shown in SEQ ID NO: 1). Part of the codons of the wild-type F gene were replaced with codons favored by Escherichia coli to obtain an optimized F gene sequence (as shown in SEQ ID NO: 2). Then 11 overlapping primers (Table 1) were designed according to the sequence of the optimized F gene for the synthesis of the optimized F gene. The total volume of PCR reaction is 25μl: 10×PCR reaction buffer 2.5μl, Primer (CF 1→CF 11, 5μmol / l) 11×1μl, 4×dNTP (10nmol / l) 1.5μl, Pfu polyrase (5U / μl) 0.5μl , ddH 2 O 9.5 μl. Denaturation at 94°C for 5min, followed by 30 cycles of 94°C for 35s, 55°C for 40s, 72°C for 40s, and finally extension at 72°C for 10min. After...
Embodiment 2
[0082] The relationship between the concentration of embodiment 2IPTG and the expression level of soluble F protein
[0083] The pET32a-F recombinant was transformed into Escherichia coli Plyss strain, and the bacteria were collected by centrifugation after being induced by 0.6mM or 1.0mM IPTG. The bacteria were ultrasonically lysed, and then centrifuged into two parts, the supernatant and the precipitate, which were boiled and loaded for SDS-PAGE electrophoresis , see the result figure 2 . A fusion protein of about 32 kDa can be seen in each sample, that is, the N-terminus of HCV F protein is fused with the C-terminus of thioredoxin of the carrier itself, which is consistent with the expected size. The density integral of the target protein induced by 0.6mM IPTG accounted for 33.4% and 18.1% ( figure 2 , lane 1 and lane 3), the density integrals of the target protein induced by 1.0mM IPTG in the corresponding samples were 34.2% and 27.1% ( figure 2 , lane 2 and lane 4). T...
Embodiment 3
[0084] Embodiment 3Ni-NTA resin purifies F protein
[0085] Collect 400ml of bacteria expressing F protein induced by IPTG by centrifugation, lyse by ultrasonic, take the supernatant and combine with Ni-NTA resin, wash the miscellaneous protein and then elute the F protein, collect the eluate of different time periods, and perform SDS-PAGE electrophoresis ,See image 3 . The eluate at different time periods contained about 32kDa F protein; the early eluate mainly contained F protein, and also contained foreign protein bands, and the gray value of the F protein band was 0.58-0.71 (lane 2 and lane 3); There was only F protein band in the eluate, no impurity protein band, and the gray value of F protein band was 0.11-0.23 (lane 4 and lane 5). The content of F protein in late eluate was determined by Bradford method, and its concentration was found to be 38-72 μg / ml. The above results showed that the content of F protein in the early eluate was higher than that in the late elua...
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