Screening and cloning for proteins transactivated by the PS1TP5 protein of hepatitis B virus: a suppression subtractive hybridization study.
OBJECTIVE
To clone and identify human genes transactivated by PS1TP5 by constructing a cDNA subtractive library with suppression subtractive hybridization (SSH) technique.


METHODS
SSH and bioinformatics techniques were used for screening and cloning of the target genes transactivated by PS1TP5 protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-myc-his(A)-PS1TP5 and pcDNA3.1(-)-myc-his(A) empty vector, respectively, and SSH technique was employed to analyze the differentially expressed DNA sequence between the two groups. After digestion with restriction enzyme Rsa I, small size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. The tester cDNA was hybridized with driver cDNA twice and subjected to nested PCR for two times, and then subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5alpha. The cDNA was sequenced and analyzed in GenBank with Vector NTI 9.1 and NCBI BLAST software after PCR amplification.


RESULTS
The subtractive library of genes transactivated by PS1TP5 was constructed successfully. The amplified library contained 90 positive clones. Colony PCR showed that 70 clones contained 200-1000-bp inserts. Sequence analysis was performed in 30 clones randomly, and the full-length sequences were obtained by bioinformatics technique. Altogether 24 coding sequences were obtained, which consisted of 23 known and 1 unknown. One novel gene with unknown functions was found and named as PS1TP5TP1 after being electronically spliced, and deposited in GenBank (accession number: DQ487761).


CONCLUSIONS
PS1TP5 is closely correlated with immunoregulation, carbohydrate metabolism, signal transduction, formation mechanism of hepatic fibrosis, and occurrence and development of tumor. Understanding PS1TP5 transactive proteins may help to bring some new clues for further studying the biological functions of pre-S1 protein.
Zhang
Jian-Kang
JK
Department of Gastroenterology, The First Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi Province, China. yeszjk@126.com
Zhao
Long-Feng
LF
Cheng
Jun
J
Guo
Jiang
J
Wang
Dan-Qiong
DQ
Hong
Yuan
Y
Mao
Yu
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods*; D004279:DNA, Viral / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D006514:Hepatitis B Surface Antigens / Q000502:physiology*; D006515:Hepatitis B virus / Q000235:genetics* / Q000502:physiology*; D018197:Hepatoblastoma / Q000235:genetics / Q000503:physiopathology; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000503:physiopathology; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011498:Protein Precursors / Q000502:physiology*; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
C519991:C6orf89 protein, human; D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; D011506:Proteins; D012333:RNA, Messenger; C050806:presurface protein 1, hepatitis B surface antigen