The Construction Of The Expression Vectors For Different RhD Transcripts

Objective: Some missplicing RhD transcripts were observed. This study aimed onthe construct of the expression vectors for normal RhD mRNA, and the missplicingtranscripts DEL9and DEL89, the sequences of which are most close to the normal RhDmRNA.Methods: Rh(D)-positive umbilical blood samples were collected from thenewborns. The Rh D, C, c, E and e antigens were all serotyped and the RHD wasgenotyped. DNA and the total RNA was extracted through the PureGene Blood Kits.The RhD transcripts including normal RHDcDNA, DEL9and DEL89were obtainedthrough both one-step RT-PCR and two-step RT-PCR, respectively. The RT-PCRproducts were purified using a Gel Extraction Kit. The normal RhD transcript obtainedthrough the two-step RT-PCR was cloned into a TOPO TA sequencing vectorpCR4-TOPO. And the RHD gene was amplified and sequenced for confirmation. Thenthe whole length RHD mRNA was subcloned into a pcDNA3.1/V5-His TOPOexpression vector. The DEL9and DEL89trscripts were cloned into the expressionvectors directly. At last, the directions and sequences of the aim genes were identified.Results: Two samples were serotyped CcDEe and CCDee respectively. PCRrevealed both samples containing RHD gene which has10exons. The total RNA wassuccessfully extracted. Total5RhD transcripts were amplified by one-step RT-PCR,and were shown by electrophoresis. The normal RHDcDNA amplified by two-stepRT-PCR was collected and purified through the Gel Extraction Kit, and wassuccessfully cloned into a sequencing vector and identified by sequencing. The DEL9and DEL89transcripts cound not be obtained from RT-PCR but were synthetised. Thenthe RHD cDNA, DEL9and DEL89were all successfully subcloned into the expression vectors. The sequences and directions were identified correctly. Thus all3expressionvectors were correctly constructed.Conclusions: The constructions of the expression vectors of the different RHDtranscripts would be important for more exploration on the red blood cell membranousprotein of RhD. And the expression vector constrction of DEL9and DEL89would besignificant to a further exploring on the membranous protein of the Delphenotype.