Construction and characterization of a cDNA library from human liver tissue with chronic hepatitis B.
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 lambdaTriplEx2 vector. Then lambda 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 x 10(6) pfu/ml and 1.49 x 10(9) 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%.


CONCLUSIONS
A high quality cDNA library from human liver tissue with chronic hepatitis B was successfully constructed.
Chen
Xiao-hong
XH
Institute of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China. Catherine89034@zju.edu.cn
Chen
Zhi
Z
Yao
Hang-ping
HP
Chen
Feng
F
Zhu
Hai-hong
HH
Zhou
Hong-juan
HJ
D018076:DNA, Complementary / Q000235:genetics*; D015723:Gene Library; D019694:Hepatitis B, Chronic / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000378:metabolism* / Q000473:pathology*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis / Q000235:genetics
D018076:DNA, Complementary; D012313:RNA