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Isolation of novel sequences targeting highly variable viral protein hemagglutinin.

Xu Z, Wu J, Feng F, Zhang X, Ma X, Tang M, Huang Y, Zhang Y, Cao Y, Cao W, He R, Gao Y, Liu Q - MethodsX (2015)

Bottom Line: Rapid evolution is a hallmark of the viral kingdom and a major concern for developing universal vaccines.The isolation of substantial numbers of viral sequence variants at highly variable viral protein domains remains a major challenge.Here we present a modification of that method in its application in the randomization of the hemagglutinin gene from a H5N2 virus, namely: •removal of potentially stressful region which harbored a stretch of basic amino acids to increase the success rates of gene cloning, and to streamline the process of future engineering of novel viral variants.•clustered randomization in a full-length gene, as the positive rate for partial gene fragment libraries was extremely low before enrichment in the previous FMDV studies.•the use of fusion partner was avoided, which was used previously for protein expression, stabilization of clones and reduction of stresses on host cells.•the use of Poisson distribution is proposed to approximate sequencing output to achieve cost effectiveness.

View Article: PubMed Central - PubMed

Affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.

ABSTRACT
Rapid evolution is a hallmark of the viral kingdom and a major concern for developing universal vaccines. The isolation of substantial numbers of viral sequence variants at highly variable viral protein domains remains a major challenge. We previously developed a combinatorial method for the isolation of novel sequences to cope with rapid viral variations at the G-H loop of Foot and Mouth Disease virus VP1 protein [1]. Here we present a modification of that method in its application in the randomization of the hemagglutinin gene from a H5N2 virus, namely: •removal of potentially stressful region which harbored a stretch of basic amino acids to increase the success rates of gene cloning, and to streamline the process of future engineering of novel viral variants.•clustered randomization in a full-length gene, as the positive rate for partial gene fragment libraries was extremely low before enrichment in the previous FMDV studies.•the use of fusion partner was avoided, which was used previously for protein expression, stabilization of clones and reduction of stresses on host cells.•the use of Poisson distribution is proposed to approximate sequencing output to achieve cost effectiveness.

No MeSH data available.


Related in: MedlinePlus

HA gene assembly via PCR amplifications. DNA fragments of different sizes were generated in a stepwise fashion, and assembled via overlapping PCR.
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fig0010: HA gene assembly via PCR amplifications. DNA fragments of different sizes were generated in a stepwise fashion, and assembled via overlapping PCR.

Mentions: The full-length gene was assembled in a stepwise fashion (Fig. 2). Two microliters of oligo 2 and oligo 3 were assembled in a 20-μl reaction using PrimeSTAR DNA polymerase in the presence of 2.5% 2-pyrrolidone [4]. The PCR mixture was heated at 94 °C for 2 min, followed by 4 cycles of amplification at 94 °C for 30 s, 33 °C for 60 s and 72 °C for 20 s, and a final extension at 72 °C for 3 min, which was then placed at 4 °C for 1 h. Three microliters of oligo 1 and 2 μl of oligo 3 as well as 0.5 μl of PrimeSTAR DNA polymerase were then added. Subsequently the PCR mixture was heated at 94 °C for 2 min, followed by 15 cycles of incubation at 94 °C for 30 s, 33 °C for 60 s and 72 °C for 20 s, and a final extension at 72 °C for 3 min, which gave rise to a 127 bp subassembly product.


Isolation of novel sequences targeting highly variable viral protein hemagglutinin.

Xu Z, Wu J, Feng F, Zhang X, Ma X, Tang M, Huang Y, Zhang Y, Cao Y, Cao W, He R, Gao Y, Liu Q - MethodsX (2015)

HA gene assembly via PCR amplifications. DNA fragments of different sizes were generated in a stepwise fashion, and assembled via overlapping PCR.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487337&req=5

fig0010: HA gene assembly via PCR amplifications. DNA fragments of different sizes were generated in a stepwise fashion, and assembled via overlapping PCR.
Mentions: The full-length gene was assembled in a stepwise fashion (Fig. 2). Two microliters of oligo 2 and oligo 3 were assembled in a 20-μl reaction using PrimeSTAR DNA polymerase in the presence of 2.5% 2-pyrrolidone [4]. The PCR mixture was heated at 94 °C for 2 min, followed by 4 cycles of amplification at 94 °C for 30 s, 33 °C for 60 s and 72 °C for 20 s, and a final extension at 72 °C for 3 min, which was then placed at 4 °C for 1 h. Three microliters of oligo 1 and 2 μl of oligo 3 as well as 0.5 μl of PrimeSTAR DNA polymerase were then added. Subsequently the PCR mixture was heated at 94 °C for 2 min, followed by 15 cycles of incubation at 94 °C for 30 s, 33 °C for 60 s and 72 °C for 20 s, and a final extension at 72 °C for 3 min, which gave rise to a 127 bp subassembly product.

Bottom Line: Rapid evolution is a hallmark of the viral kingdom and a major concern for developing universal vaccines.The isolation of substantial numbers of viral sequence variants at highly variable viral protein domains remains a major challenge.Here we present a modification of that method in its application in the randomization of the hemagglutinin gene from a H5N2 virus, namely: •removal of potentially stressful region which harbored a stretch of basic amino acids to increase the success rates of gene cloning, and to streamline the process of future engineering of novel viral variants.•clustered randomization in a full-length gene, as the positive rate for partial gene fragment libraries was extremely low before enrichment in the previous FMDV studies.•the use of fusion partner was avoided, which was used previously for protein expression, stabilization of clones and reduction of stresses on host cells.•the use of Poisson distribution is proposed to approximate sequencing output to achieve cost effectiveness.

View Article: PubMed Central - PubMed

Affiliation: MOE Key Laboratory of Aquatic Product Safety, State Key Laboratory of Biocontrol, Biotechnology Research Center, The School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.

ABSTRACT
Rapid evolution is a hallmark of the viral kingdom and a major concern for developing universal vaccines. The isolation of substantial numbers of viral sequence variants at highly variable viral protein domains remains a major challenge. We previously developed a combinatorial method for the isolation of novel sequences to cope with rapid viral variations at the G-H loop of Foot and Mouth Disease virus VP1 protein [1]. Here we present a modification of that method in its application in the randomization of the hemagglutinin gene from a H5N2 virus, namely: •removal of potentially stressful region which harbored a stretch of basic amino acids to increase the success rates of gene cloning, and to streamline the process of future engineering of novel viral variants.•clustered randomization in a full-length gene, as the positive rate for partial gene fragment libraries was extremely low before enrichment in the previous FMDV studies.•the use of fusion partner was avoided, which was used previously for protein expression, stabilization of clones and reduction of stresses on host cells.•the use of Poisson distribution is proposed to approximate sequencing output to achieve cost effectiveness.

No MeSH data available.


Related in: MedlinePlus