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Journal of Zhejiang University SCIENCE B 2005 Vol.6 No.4 P.288~294

http://doi.org/10.1631/jzus.2005.B0288


Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B


Author(s):  CHEN Xiao-hong, CHEN Zhi, YAO Hang-ping, CHEN Feng, ZHU Hai-hong, ZHOU Hong-juan

Affiliation(s):  Institute of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China; more

Corresponding email(s):   Catherine89034@zju.edu.cn

Key Words:  cDNA library, Human liver tissue, Chronic hepatitis B, Construction and characterization


CHEN Xiao-hong, CHEN Zhi, YAO Hang-ping, CHEN Feng, ZHU Hai-hong, ZHOU Hong-juan. Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B[J]. Journal of Zhejiang University Science B, 2005, 6(4): 288~294.

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author="CHEN Xiao-hong, CHEN Zhi, YAO Hang-ping, CHEN Feng, ZHU Hai-hong, ZHOU Hong-juan",
journal="Journal of Zhejiang University Science B",
volume="6",
number="4",
pages="288~294",
year="2005",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.2005.B0288"
}

%0 Journal Article
%T Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B
%A CHEN Xiao-hong
%A CHEN Zhi
%A YAO Hang-ping
%A CHEN Feng
%A ZHU Hai-hong
%A ZHOU Hong-juan
%J Journal of Zhejiang University SCIENCE B
%V 6
%N 4
%P 288~294
%@ 1673-1581
%D 2005
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.2005.B0288

TY - JOUR
T1 - Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B
A1 - CHEN Xiao-hong
A1 - CHEN Zhi
A1 - YAO Hang-ping
A1 - CHEN Feng
A1 - ZHU Hai-hong
A1 - ZHOU Hong-juan
J0 - Journal of Zhejiang University Science B
VL - 6
IS - 4
SP - 288
EP - 294
%@ 1673-1581
Y1 - 2005
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.2005.B0288


Abstract: 
Objective: To construct a cDNA library from human liver tissue with chronic hepatitis B and check its quality for investigating the expression level of liver tissue infected by hepatitis B virus. This will then be used to find the relevant genes and interesting proteins associated with the development of hepatitis B. Methods: The total RNA from liver tissue with chronic hepatitis B was extracted and the mRNA was purified using TRIZOL method. Switching mechanism at 5′ end of the RNA transcript (SMART) technique and CDS III/3′ primer were used for first-strand cDNA synthesis. Long distance polymerase chain reaction (LD PCR) was then used to synthesize the double-strand cDNA that was then digested by Sfi I and fractionated by CHROMA SPIN-400 column. The longer than 0.4 kb cDNAs were collected and ligated to λTriplEx2 vector. Then λ phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. Fourteen plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. Results: The titers of unamplifed and amplified libraries were 1.94×106 pfu/ml and 1.49×109 pfu/ml respectively. The percentages of recombinants from both libraries were 98.15% in unamplified library and 98.76% in amplified library. The lengths of the inserts were 1.23 kb in average, 1-2 kb in 64.29%, and 0.5-1.0 kb in 35.71%. Conclusion: A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.

Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article

Reference

[1] Barnes, W.M., 1994. PCR amplification of up to 35-kb DNA with high fidelity and high yield from λ bacteriophage templates. Proc. Natl. Acad. Sci., 91(6):2216-2220.

[2] Chin, R., Locarnini, S., 2003. Treatment of chronic hepatitis B: current challenges and future directions. Rev Med Viro, 13(4):255-272.

[3] Forti, S., Scanlan, M.J., Invernizzi, A., Castiglioni, F., Pupa, S., Agresti, R., Fontancelli, R., Morelli, D., Old, L.J., Pupa, S.M., Menard, S., 2002. Identification of breast cancer-restricted antigens by antibody screening of SKBR3 cDNA library using a preselected patient’s serum. Breast Cancer Res Treat, 73(3):245-256.

[4] Ganem, D., Prince, A.M., 2004. Hepatitis B virus infection(natural history and clinical consequences. N Engl J Med., 350(11):1118-1129.

[5] Huarte, M., Sanz-Ezquerro, J.J., Roncal, F., Ortin, J., Nieto, A., 2001. PA subunit from influenza virus polymerase complex interacts with a cellular protein with homology to a family of transcriptional activators. J Virol, 75(18):8597-8604.

[6] Liaw, Y.F., Tai, D.I., Chu, C.M., Chen, T.J., 1988. The development of cirrhosis in patients with chronic type B hepatitis: a prospective study. Hepatology, 8(3):493-496.

[7] Myers, E.L., Allen, J.F., 2002. Tsg101, an inactive homologue of ubiquitin ligase e2, interacts specifically with human immunodeficiency virus type 2 gag polyprotein and results in increased levels of ubiquitinated gag. J Virol, 76(22):11226-11235.

[8] Weiskirchen, R., Moser, M., Weiskirchen, S., Erdel, M., Dahmen, S., Buettner, R., Gressner, A.M., 2001. LIM-domain protein cysteine and glycine-rich protein 2 (CRP2) is a novel marker of hepatic stellate cells and binding partner of the protein inhibitor of activated STAT1. Biochem J, 359(3):485-496.

[9] Wright, K.O., Messing, E.M., Reeder, J.E., 2002. Increased expression of the acid sphingomyelinase-like protein ASML3a in bladder tumors. J Urol, 168(6):2645-2649.

[10] Sambrook, J., Russell, D.W., 2002. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Lab Press, p.11.8, 11.26-11.35.

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