Mutant of hemagglutinin protein of h3n2 subtype influenza virus and its application
A hemagglutinin protein, influenza virus technology, applied in the field of H3N2 subtype influenza virus hemagglutinin protein mutant, infection and/or diseases caused by the infection, can solve complex post-translational glycosyl modifications, research Significant progress has not yet been achieved to achieve the effect of not easy to quickly fail, overcoming the loss of immune efficacy, and ideal immune effect
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Embodiment 1
[0157] Example 1: Preparation of H3N2 influenza virus HA protein and its mutants
[0158] (a) Design and structure of HA protein mutants
[0159] In the native HA protein of influenza virus, the amino acid that undergoes N-linked glycosylation is usually asparagine (N) in the characteristic sequence N-X-(S or T), where N represents asparagine and X represents except proline Any amino acid other than acid, S stands for serine and T stands for threonine. In this example, N-linked glycosylation of HA protein was removed by mutating asparagine (N) to alanine (A) in the characteristic sequence N-X- (S or T) in native HA protein site.
[0160] The native HA protein (WI2005-WT-HA) used in this example was derived from the HA protein of H3N2 subtype influenza virus strain A / Wisconsin / 67 / 2005. The HA protein of the strain comprises the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acids 1-10 of SEQ ID NO: 1 are the signal peptide, the amino acids 504-550 are the trans...
Embodiment 2
[0179] Example 2: Polyacrylamide gel electrophoresis (SDS-PAGE) analysis
[0180] The six proteins prepared in Example 1 (native HA protein, HA-mut1, HA-mut2, HA-mut3, HAmg and HAug) were analyzed using polyacrylamide gel electrophoresis (SDS-PAGE). The top gel used was a 5% stacking gel (prepared as follows: 830 μl of 30% acrylamide, 630 μl of 1M Tris (pH 6.8), 50 μl of 10% SDS, 50 μl of 10% ammonium persulfate and 5 μl TEMED). The underlying gel used was a 12% resolving gel (prepared as follows: to 3.3 ml of water was added 4 ml of 30% acrylamide, 2.5 ml of 1M Tris (pH 8.8), 100 μl of 10% SDS, 100 μl of 10% ammonium persulfate and 10 μl of TEMED). The electrophoresis conditions used were electrophoresis at 150V for 2 hours. After electrophoresis, polyacrylamide gels were stained with Coomassie brilliant blue (Sigma). The experimental results are as image 3 shown.
[0181] image 3 The SDS-PAGE analysis results of the six proteins prepared in Example 1 are shown; wher...
Embodiment 3
[0182] Example 3: Evaluation of neutralizing activity of antiserum
[0183] (a) Immunoassay
[0184]6-week-old, SPF grade, female Balb / C mice were provided by the Experimental Animal Center of Xiamen University, weighing approximately 20 g. The six proteins prepared in Example 1 (native HA protein, HA-mut1, HA-mut2, HA-mut3, HAmg and HAug) and PBS (used as a negative control) were respectively mixed with aluminum adjuvant in a volume of 1:1 than mixed for immunization of mice. The immunization protocol was as follows: 6 mice were used in each group, the immunization method was intramuscular injection, the immunization dose was 5 μg protein / mouse, and the injection volume was 100 μl / mouse. The immunization was performed twice, and the interval between the two immunizations was 14 days. . Mouse sera were collected 14 days after the second immunization. The collected serum samples were inactivated at 56°C for 30 minutes, and then stored at -20°C for later use.
[0185] (b) E...
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