1. 懷孕檢驗(Pregnancy Testing)

雖然測定血清或尿液中得hCG都可用來確定女性是否懷孕，然而一般的懷孕試驗大都採用尿液為測定檢體。

　

2. 異位懷孕(Ectopic Pregnancy)

測定血清中hCG中的濃度，有助於診斷異位懷孕或子宮外孕。

　

3. 威脅性流產(Threatened Abortion)

在妊娠期間，若有子宮出血，子宮頸變軟而擴大等徵狀，可能導致流產，稱為”威脅性流產(threatened abortion)”。

　

4. 滋養層腫瘤(Trophoblastic Tumors)

1. α、β次單元的cDNA clones

將RNA從胎盤組織中用Phenol-Chloroform萃取出來，用酒精沉澱RNA，通過dT-cellulose以增多poly A mRNA，最好占全部mRNA的2%，接著做反轉錄得到胎盤的cDNA 庫。質體用pBR322內含PstI限制酵素切割位置，將此重組質體transform到E.coli 細胞以建立cDNA 庫。

　

2. 建構質體p α SVHVPI

以pBR322為基礎

Referring to FIG. 1, in order to construct the plasmid alpha 970 H/B, a cDNA clone containing the alpha hCG fragment is digested with NcoI. The NcoI site, just 5' to the ATG codon signalling initiation of translation, is filled in and ligated to a synthetic HindIII linker. Similarly, the natural HindIII site in the 3' untranslated region of the clone is cut, filled in with E. coli DNA polymerase Klenow, and then ligated to a synthetic BamHI linker. This fragment is cloned into the plasmid pBR322 between its HindIII and BamHI sites to generate the plasmid alpha 574 H/B. This plasmid is digested with BamHI,treated with alkaline phosphatase, and ligated to the 396 bp Sau3A fragment of SV40 DNA (from 0.07 to 0.14 map units) which has been isolated from a polyacrylamide gel. The ligation mix is used to transform E. coli to ampicillin resistance and the desired plasmid, alpha 970 H/B, is identified among the transformants. The plasmid Q(2) 7 is constructed by cutting SV40 at its HsaII site, making flush ends by digestion with nuclease S1, ligating on EcoRI linkers, digesting with EcoRI, and cloning the resulting 1436 bp fragment into the EcoRI site of pBR322. Referring to FIG. 1, Q(2) 7 is digested completely with EcoRI and partially with HindIII; the fragment from 0.72 to 0.94 map units is isolated and cloned into alpha 970 H/B, which has been digested with ScoRI and HindIII and treated with alkaline phosphatase. The ligation mix is used to transform E. coli, and the desired plasmid, p alpha SVL, is identified among the transformants by restriction mapping. p alpha SVL is digested with EcoRI and the fragment of SV40, with EcoRI ends, extending from 0 to 0.72 map units, and containing the SV40 origin of replication and the intact early region, is ligated to it to generate the plasmid p alpha SVHVPl, which is isolated from E. coli transformants.

　

3. 建構質體p β SVVP1

以pBR322為基礎

A 579 bp cDNA clone coding for beta hCG was obtained from John C. Fiddes at Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (Fiddes et al, Nature, vol. 286, pp. 684-687 (1980)). This fragment is ligated at each end to synthetic BamHI linkers. After digestion by HgaI restriction enzyme, the ends are filled in with Klenow DNA polymerase and synthetic EcoRI linkers are ligated on so that an EcoRI site is about 10 bp 5' to the ATG codon of the signal peptide coding sequence. A BamHI site is about 60 bp 3' to the nonsense codon marking the end of the coding sequence. Referring to FIG. 2, this 556 bp EcoRI-BamHI fragment is isolated and cloned into pBR322, between the EcoRI and BamHI sites, to give the plasmid p beta 556 R/B. In order to construct the plasmid pSVHR (FIG. 2), SV40 DNA is partially digested with HindIII to yield linear molecules, digested with nuclease S1 to make flush ends, ligated to synthetic EcoRI linkers and digested with EcoRI and BamHI. The fragment from 0.94 to 0.14 map units, containing the SV40 origin of replication and early region, is cloned into pBR322 as an EcoRI-BamHI piece. Referring still to FIG. 2, the EcoRI site of the plasmid p beta 556 R/B is methylated in a reaction catalyzed by EcoRI methylase, following which the plasmid is cut with NdeI. EcoRI linkers are ligated to the S1 treated NdeI flush ends and activated by digestion with EcoRI, which is followed by digestion with BamHI. The SV40 fragment of pSVHR from the EcoRI site to the BamHI site is isolated and ligated in a reaction mix containing the digestion fragments of p beta 556 R/B. Following ligation, the mix is digested with SalI to eliminate plasmids which have re-inserted the EcoRI (NdeI) to BamHI piece of pBR322. E. coli is transformed with the digested ligation mix and p beta SVVPl is identified and isolated.

　

4. 建構質體p αβ SVVP1

　

Referring to FIG. 3, pBR322/Kpn is derived from pBR322 by inserting a KpnI linker into its unique EcoRI site, after this site is deleted by digestion with EcoRI, followed by digestion with S1 nuclease. Referring still to FIG. 3, SV40 DNA is digested with AvaII. The staggered ends of the resulting fragments are filled in by Klenow DNA polymerase to form flush ends, and the mixture is then fractionated on a polyacrylamide gel. The 682 base pair fragment (0.64 to 0.77 map units) containing the origin of replication and to the unique KpnI site is isolated from the gel, ligated to synthetic HindIII linkers, and digested with HindIII and KsnI. The resulting fragments are ligated to pBR322/Kpn. p266, which contains the 266 base pair KpnI HindIII fragment, including the SV40 late promoter region, is isolated. p266 is cut with HindIII and BamHI, and treated with bacterial alkaline phosphatase. Still referring to FIG. 3, p beta SVVPI/B is constructed as follows: p beta SVVPI (FIG. 2) is cut with EcoRI, followed by ligation to eliminate pBR322 sequences. Subsequently, this DNA is cut with BamHI and cloned into the BamHI site of pBR322. The resulting plasmid, p beta SVVPI/B, is then digested with HindIII and BamHI and the 1003 base pair HindIII-BamHI fragment is ligated into p266 to yield the plasmid p beta VPl 266, in which the beta hCG cDNA is positioned downstream from the SV40 late promoter in such a way that its RNA transcript would be spliced as if it were the viral VP1 transcript. The alpha hCG cDNA is inserted into p beta VPl 266 as a HindIII fragment, which has been cut at its HindIII site and treated with bacterial alkaline phosphatase. E. coli transfor-

mants derived from this ligation are screened by restriction mapping, and plasmids are isolated that have the desired structure, in which the alpha hCG cDNA has replaced VP2 in the correct orientation, followed downstream by the beta hCG cDNA, which has replaced VP1. One such isolated plasmid, p alpha beta VP1, is used to complete the construction of p alpha beta SVVPl. The plasmid is cut with KpnI, and the full SV40 genome, cut with KpnI, is inserted by ligation into this site. Following transformation of E. coli, a plasmid with the required structure, p alpha beta SVVPl, is isolated. This plasmid contains DNA encoding both the alpha and beta subunits of hCG, and thus is capable of directing the expression, in host mammalian cells, of both subunits, whereby biologically functional, glycosylated heterodimeric hCG is produced (glycosylation occurs post translationally).

　

5. 建構質體p RF375、pRF398及pRF398 alpha t(2)

　

Referring to FIG. 4, the plasmid CL28 (identical to plasmid JYMMT(E); Haner et al., J. Mol. Applied Gen., 1, 273-288 (1983)), containing the murine metallothionein promoter, SV40 DNA, and pBR322 sequences, is cut with the restriction endonuclease Bgl II. At this site are inserted cDNA clones of either alpha hCG or beta hCG, containing untranslated regions of about 10 and 30 bp at their 5' and of about 220 and 60 bp at their 3' ends. These clones have been genetically engineered by the addition of synthetic BamHI linkers at their termini. The resulting plasmids pRF 302 (alpha) or pRF 394 (beta) are digested with restriction enzymes BamHI and SalI to release the SV40 DNA sequences. Plasmid pB2-2, which contains the entire BPV genome, and some pBR322 sequences, is digested with BamHI and SalI to yield the BPV genome with BamHI/SalI ends; this fragment is ligated into pRF 302 (alpha) and pRF 394 (beta) containing the metallothionein-hCG sequences. Following transformation of E. coli, plasmids pRF 375 and pRF 398 are identified and isolated. They encode alpha hCG or beta hCG, respectively, under the control of the mouse metallothionein promoter. Construction of the Plasmid RF 398 alpha t(2) Referring to FIG. 5, the plasmid p alpha t(2) is derived by cloning the alpha hCG 574 HindIII fragment into plasmid pVBt2 (V. B. Reddy et al., PNAS, 79, 2064-2067, 1982). p alpha t(2), which contains the alpha hCG cDNA under the control of the SV40 early promoter, is digested with EcoRI. The 5' overhangs are removed by S1 nuclease digestion prior to the addition of synthetic BamHI linkers by blunt end ligation. Plasmid RF 398 (FIG. 4) is digested with BamHI and treated with bacterial alkaline phosphatase. The 1735 base pair BamHI fragment of p alpha t(2) is inserted in to RF 398. The resulting plasmid RF 398 alpha t(2) is isolated from E. coli transformants. This plasmid thus has the beta hCG cDNA in a transcriptional unit under control of the mouse metallothionein promoter and the alpha hCG cDNA in a transcriptional unit controlled by the SV40 early promoter.

　

　

6. 將表現載體transfect到老鼠細胞株C127 cell line

Genezyme corporation用mouse cell line作表現，可以得到生物活性很高的重組hCG，但是該公司目前並沒有相關應用產品推出。此外用酵母菌 Pichia pastoris表現亦可得到生物活性很高的重組hCG，以及很高的表現量24mg/l 的α次單元及2.7mg/l的β次單元在shake culture中，預期在bioreactor中會得到更多(Sen Gupta., et.al. 1999)。