Molecular Cloning, Bioinformatics Analysis and Expression of Insulin-Like Growth Factor 2 from Tianzhu White Yak, Bos grunniens
Abstract
:1. Introduction
2. Results and Discussion
2.1. Result
2.1.1. Cloning and Characterization of the Full Coding Region of the Yak IGF2 Gene
2.1.2. Amino Acid Composition and Protein Secondary Structure
2.1.3. Multiple Sequence Alignment
2.1.4. 3D Structure Prediction
2.1.5. Similarities between Structure of Yak IGF2 and Other Mammalian IGF2
2.1.6. Expression of IGF2 Gene by Real Time PCR in Tianzhu White Yak
2.2. Discussion
3. Experimental Section
3.1. Brief Introduction of Methods
3.2. Samples and Materials
3.3. RNA Isolation and cDNA Synthesis
3.4. Synthesis and Confirmation of Partial cDNA of IGF2 Gene
3.5. Rapid Amplification of cDNA Ends (RACE), Cloning and Sequencing
3.6. Analysis and Alignment of cDNA Sequence
3.7. Protein and mRNA Secondary Structure Prediction
3.8. Multiple Sequence Alignment and Phylogenetic Analysis
3.9. Yak IGF2 Protein 3D Structure Prediction
3.10. Real Time PCR Assays for IGF2 Gene Expression in Yak
4. Conclusions
Acknowledgments
Conflicts of Interest
References
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Amimo Acid | Number Count | % By Weight | % By Frequency |
---|---|---|---|
Ala (A) | 20 | 7.22 | 11.17 |
Arg (R) | 18 | 14.28 | 10.06 |
Asn (N) | 1 | 0.58 | 0.56 |
Asp (D) | 9 | 5.26 | 5.03 |
Cys (C) | 8 | 4.19 | 4.47 |
Gln (Q) | 5 | 3.26 | 2.79 |
Glu (E) | 9 | 5.90 | 5.03 |
Gly (G) | 12 | 3.48 | 6.70 |
His (H) | 2 | 1.39 | 1.12 |
Ile (I) | 5 | 2.87 | 2.79 |
Pyl (O) | 0 | 0 | 0 |
Leu (L) | 19 | 10.92 | 10.61 |
Lys (K) | 7 | 4.56 | 3.91 |
Met(M) | 1 | 0.67 | 0.56 |
Phe (F) | 9 | 6.73 | 5.03 |
Pro (P) | 9 | 4.44 | 5.03 |
Ser (S) | 17 | 7.52 | 9.50 |
Thr (T) | 12 | 6.16 | 6.70 |
Trp (W) | 1 | 0.95 | 0.56 |
Tyr (Y) | 6 | 4.97 | 3.35 |
Val (V) | 9 | 4.53 | 5.03 |
Sec (U) | 0 | 0 | 0 |
IGF-2 | (NCBI Reference Sequence) | No. of Residues | Total Score | Coverage (%) | Identity (%) | Positive (%) | E-Value | pI | Mean Value | Gaps (%) |
---|---|---|---|---|---|---|---|---|---|---|
Bos grunniens | KF682139 | 179 | 364 | 100 | 100 | 100 | 2e- 134 | 8.82 | 19.68 | 0 |
Bos taurus | AY957981.1 | 179 | 362 | 100 | 99 | 99 | 4e- 133 | 8.65 | 19.64 | 0 |
Bubalus bubalis | KC107769.1 | 135 | 278 | 75 | 100 | 100 | 3e- 101 | 9.23 | 13.77 | 0 |
Bos primigenius | AF283002.1 | 149 | 305 | 83 | 100 | 100 | 2e- 111 | 10.50 | 15.11 | 0 |
Ovis aries | M89788.1 | 179 | 331 | 100 | 96 | 97 | 5e -121 | 8.49 | 19.62 | 0 |
Capra hircus | DQ645739.1 | 179 | 350 | 100 | 96 | 97 | 9e -129 | 8.49 | 19.65 | 0 |
Cervus elaphus | EF177491.1 | 179 | 335 | 100 | 92 | 94 | 1e -122 | 8.67 | 19.72 | 0 |
Sus scrofa | HQ450757.1 | 181 | 289 | 98 | 84 | 88 | 2e -104 | 9.99 | 20.31 | 0 |
Homo sapiens | XM005252900.1 | 180 | 309 | 100 | 84 | 88 | 1e -112 | 9.34 | 20.14 | 0 |
Structure | (PDB) | No. of Residues | Aligned Residues | RMSD | Q-Score | p-Score | Z-Score |
---|---|---|---|---|---|---|---|
Predicted yak IGF2 | human IGF1 (1H02) | 64 | 49 | 1.61 | 0.45 | 1.8 | 3.8 |
Predicted yak IGF2 | human IGF2 (1IGL) | 67 | 65 | 0.09 | 0.97 | 10.8 | 9.7 |
NO. | Start Position | End Position | Peptide | Peptide Length |
---|---|---|---|---|
1 | 6 | 27 | GKSVLVLLAFLAFSCCYAAYR | 22 |
2 | 29 | 48 | SETLCGGELVDTLQFVCGDR | 20 |
3 | 67 | 87 | VEECCFRSCDLALLETYCATP | 21 |
4 | 93 | 119 | DVSASTTVLPDDVTAYPVGKFFQYDIW | 27 |
5 | 127 | 135 | RRGLPAFLR | 9 |
6 | 155 | 164 | HRPLIALPTQ | 10 |
Function | Software and website |
---|---|
To search nucleotide sequence | http://www.ncbi.nlm.nih.gov/nuccore/ [29] |
To find conserved sequence | MEGA 5.1/DNAstar/DNAMAN [30] |
To design primers in the conserved sequence | Oligo 7.0/Primer Premier 5 [31,32] |
To find open reading frame | http://www.ncbi.nlm.nih.gov/gorf/gorf.html [33] |
To analysis amino sequence | PROTEAN program/DNAMAN [17,30] |
To analysis identity of amino acid | http://blast.ncbi.nlm.nih.gov/Blast.cgi [34] |
To alignmultiple sequence | MAFFT Multiple program/Sequence Alignment [18] |
To analysis phylogenetic | MAFFT Multiple program/Sequence Alignment/MEGA 5.1 [18] |
To predict secondary structure | Bioinformatics Toolkit Quick2D program [35] |
To predict 3D structure | http://swissmodel.expasy.org/ [20] |
To analysis 3D structure | Swiss-PDB Viewer program [20] |
To predict antigenic properties | http://imed.med.ucm.es/Tools/antigenic.pl [36] |
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Zhang, Q.; Gong, J.; Wang, X.; Wu, X.; Li, Y.; Ma, Y.; Zhang, Y.; Zhao, X. Molecular Cloning, Bioinformatics Analysis and Expression of Insulin-Like Growth Factor 2 from Tianzhu White Yak, Bos grunniens. Int. J. Mol. Sci. 2014, 15, 504-524. https://doi.org/10.3390/ijms15010504
Zhang Q, Gong J, Wang X, Wu X, Li Y, Ma Y, Zhang Y, Zhao X. Molecular Cloning, Bioinformatics Analysis and Expression of Insulin-Like Growth Factor 2 from Tianzhu White Yak, Bos grunniens. International Journal of Molecular Sciences. 2014; 15(1):504-524. https://doi.org/10.3390/ijms15010504
Chicago/Turabian StyleZhang, Quanwei, Jishang Gong, Xueying Wang, Xiaohu Wu, Yalan Li, Youji Ma, Yong Zhang, and Xingxu Zhao. 2014. "Molecular Cloning, Bioinformatics Analysis and Expression of Insulin-Like Growth Factor 2 from Tianzhu White Yak, Bos grunniens" International Journal of Molecular Sciences 15, no. 1: 504-524. https://doi.org/10.3390/ijms15010504